Polypeptides Homologous To VEGF and BMP1

ABSTRACT

The present invention involves the identification and preparation of vascular endothelial growth factor-E (VEGF-E). VEGF-E is a novel polypeptide related to vascular endothelial growth factor (VEGF) and bone morphogenetic protein 1. VEGF-E has homology to VEGF including conservation of the amino acids required for activity of VEGF. VEGF-E can be useful in wound repair, as well as in the generation and regeneration of tissue.

RELATED APPLICATIONS

This application is a continuation of pending U.S. application Ser. No.10/863,133 filed on Jun. 8, 2004, which is a divisional of pending U.S.application Ser. No. 10/178,442, filed on Jun. 19, 2002, which is adivisional of U.S. application Ser. No. 09/265,686 filed Mar. 10, 1999,now U.S. Pat. No. 6,455,283, which is a continuation-in-part of U.S.application Ser. No. 09/184,216 filed Nov. 2, 1998, now abandoned, whichis a continuation-in-part of U.S. application Ser. No. 09/040,220, filedMar. 17, 1998, now U.S. Pat. No. 6,391,311, the disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to polypeptides related to vascularendothelial cell growth factor (hereinafter sometimes referred to asVEGF) and bone morphogenetic protein 1 (hereinafter sometimes referredto as BMP1), termed herein as VEGF-E polypeptides, nucleic acidsencoding therefor, methods for preparing VEGF-E, and methods,compositions, and assays utilizing VEGF-E.

BACKGROUND OF THE INVENTION

Various naturally occurring polypeptides reportedly induce theproliferation of endothelial cells. Among those polypeptides are thebasic and acidic fibroblast growth factors (FGF) (Burgess and Maciag,Annual Rev. Biochem., 58: 575 (1989)), platelet-derived endothelial cellgrowth factor (PD-ECGF) (Ishikawa et al., Nature, 338: 557 (1989)), andvascular endothelial growth factor (VEGF). Leung et al., Science, 246:1306 (1989); Ferrara and Henzel, Biochem. Biophys. Res. Commun., 161:851 (1989); Tischer et al., Biochem. Biophys. Res. Commun., 165: 1198(1989); EP 471,754B granted Jul. 31, 1996.

The heparin-binding endothelial cell-growth factor, VEGF, was identifiedand purified from media conditioned by bovine pituitary follicular orfolliculo-stellate cells several years ago. See Ferrara et al., Biophys.Res. Comm., 161: 851 (1989). Media conditioned by cells transfected withthe human VEGF (hVEGF) cDNA promoted the proliferation of capillaryendothelial cells, whereas control cells did not. Leung et al., Science,246: 1306 (1989). VEGF is a naturally occurring compound that isproduced in follicular or folliculo-stellate cells (FC), amorphologically well-characterized population of granular cells. The FCare stellate cells that send cytoplasmic processes between secretorycells.

VEGF is expressed in a variety of tissues as multiple homodimericisoforms (121, 165, 189 and 206 amino acids per monomer), alsocollectively referred to as hVEGF-related proteins, resulting fromalternative RNA splicing. The 121-amino acid protein differs from hVEGFby virtue of the deletion of the 44 amino acids between residues 116 and159 in hVEGF. The 189-amino acid protein differs from hVEGF by virtue ofthe insertion of 24 amino acids at residue 116 in hVEGF, and apparentlyis identical to human vascular permeability factor (hVPF). The 206-aminoacid protein differs from hVEGF by virtue of an insertion of 41 aminoacids at residue 116 in hVEGF. Houck et al., Mol. Endocrin., 5: 1806(1991); Ferrara et al., J. Cell. Biochem., 47: 211 (1991); Ferrara etal., Endocrine Reviews, 13: 18 (1992); Keck et al., Science 246: 1309(1989); Connolly et al., J. Biol. Chem., 264: 20017 (1989); EP 370,989published May 30, 1990. VEGF₁₂₁ is a soluble mitogen that does not bindheparin; the longer forms of VEGF bind heparin with progressively higheraffinity. The heparin-binding forms of VEGF can be cleaved in thecarboxy terminus by plasmin to release (a) diffusible form(s) of VEGF.The amino acid sequence of the carboxy-terminal peptide identified afterplasmin cleavage is Arg₁₁₀-Ala₁₁₁. Amino terminal “core” protein, VEGF(1-110), isolated as a homodimer, binds neutralizing monoclonalantibodies (4.6.1 and 2E3) and soluble forms of FMS-like tyrosine kinase(FLT-1), kinase domain region (KDR) and fetal liver kinase (FLK)receptors with similar affinity compared to the intact VEGF₁₆₅homodimer.

As noted, VEGF contains two domains that are responsible respectivelyfor binding to the KDR and FLT-1 receptors. These receptors exist onlyon endothelial (vascular) cells. As cells become depleted in oxygen,because of trauma and the like, VEGF production increases in such cellswhich then bind to the respective receptors in order to signal ultimatebiological effect. The signal then increases vascular permeability andthe cells divide and expand to form new vascular pathways—vasculogenesisand angiogenesis.

Thus, VEGF is useful for treating conditions in which a selected actionon the vascular endothelial cells, in the absence of excessive tissuegrowth, is important, for example, diabetic ulcers and vascular injuriesresulting from trauma such as subcutaneous wounds. Being a vascular(artery and venus) endothelial cell growth factor, VEGF restores cellsthat are damaged, a process referred to as vasculogenesis, andstimulates the formulation of new vessels, a process referred to asangiogenesis.

VEGF would also find use in the restoration of vasculature after amyocardial infarct, as well as other uses that can be deduced. In thisregard, inhibitors of VEGF are sometimes desirable, particularly tomitigate processes such as angiogenesis and vasculogenesis in cancerouscells.

It is now well established that angiogenesis, which involves theformation of new blood vessels from preexisting endothelium, isimplicated in the pathogenesis of a variety of disorders. These includesolid tumors and metastasis, atherosclerosis, retrolental fibroplasia,hemangiomas, chronic inflammation, intraocular neovascular syndromessuch as proliferative retinopathies, e.g., diabetic retinopathy,age-related macular degeneration (AMD), neovascular glaucoma, immunerejection of transplanted corneal tissue and other tissues, rheumatoidarthritis, and psoriasis. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992); Klagsbrun et al., Annu. Rev. Physiol., 53: 217-239(1991); and Garner A, “Vascular diseases”, In: Pathobiology of OcularDisease. A Dynamic Approach, Garner A, Klintworth G K, Eds., 2nd Edition(Marcel Dekker, NY, 1994), pp 1625-1710.

In the case of tumor growth, angiogenesis appears to be crucial for thetransition from hyperplasia to neoplasia, and for providing nourishmentto the growing solid tumor. Folkman et al., Nature 339: 58 (1989). Theneovascularization allows the tumor cells to acquire a growth advantageand proliferative autonomy compared to the normal cells. Accordingly, acorrelation has been observed between density of microvessels in tumorsections and patient survival in breast cancer as well as in severalother tumors. Weidner et al., N Engl J Med, 324: 1-6 (1991); Horak etal., Lancet, 340: 1120-1124 (1992); Macchiarini et al., Lancet, 340:145-146 (1992).

The search for positive regulators of angiogenesis has yielded manycandidates, including aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin,IL-8, etc. Folkman et al., J.B.C., supra, and Klagsbrun et al., supra.The negative regulators so far identified include thrombospondin (Goodet al., Proc. Natl. Acad. Sci. USA., 87: 6624-6628 (1990)), the16-kilodalton N-terminal fragment of prolactin (Clapp et al.,Endocrinology, 133: 1292-1299 (1993)), angiostatin (O'Reilly et al.Cell, 79: 315-328 (1994)), and endostatin. O'Reilly et al, Cell, 88:277-285 (1996). Work done over the last several years has establishedthe key role of VEGF, not only in stimulating vascular endothelial cellproliferation, but also in inducing vascular permeability andangiogenesis. Ferrara et al., Endocr. Rev., 18: 4-25 (1997). The findingthat the loss of even a single VEGF allele results in embryoniclethality points to an irreplaceable role played by this factor in thedevelopment and differentiation of the vascular system. Furthermore,VEGF has been shown to be a key mediator of neovascularizationassociated with tumors and intraocular disorders. Ferrara et al.,Endocr. Rev., supra. The VEGF mRNA is overexpressed by the majority ofhuman tumors examined. Berkman et al., J Clin Invest, 91: 153-159(1993); Brown et al., Human Pathol., 26: 86-91 (1995); Brown et al.,Cancer Res. 53: 4727-4735 (1993); Mattem et al., Brit. J. Cancer, 73:931-934 (1996); Dvorak et al., Am J. Pathol., 146: 1029-1039 (1995).

Also, the concentration levels of VEGF in eye fluids are highlycorrelated to the presence of active proliferation of blood vessels inpatients with diabetic and other ischemia-related retinopathies. Aielloet al., N. Engl. J. Med., 331: 1480-1487 (1994). Furthermore, recentstudies have demonstrated the localization of VEGF in choroidalneovascular membranes in patients affected by AMD. Lopez et al., Invest.Ophthalmol. Vis. Sci., 37: 855-868 (1996). Anti-VEGF neutralizingantibodies suppress the growth of a variety of human tumor cell lines innude mice (Kim et al., Nature, 362: 841-844 (1993); Warren et al., J.Clin. Invest., 95: 1789-1797 (1995); Borgstrom et al., Cancer Res. 56:4032-4039 (1996); Melnyk et al., Cancer Res. 56: 921-924 (1996)) andalso inhibit intraocular angiogenesis in models of ischemic retinaldisorders. Adamis et al., Arch. Ophthalmol., 114: 66-71 (1996).Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGFaction are promising candidates for the treatment of solid tumors andvarious intraocular neovascular disorders. Such antibodies aredescribed, for example, in EP 817,648 published Jan. 14, 1998 and inPCT/US 98/06724 filed Apr. 3, 1998.

Regarding the bone morphogenetic protein family, members of this familyhave been reported as being involved in the differentiation of cartilageand the promotion of vascularization and osteoinduction in preformedhydroxyapatite. Zou, et al., Genes Dev. (U.S.), 11(17):2191 (1997);Levine, et al., Ann. Plast. Surg., 39(2):158 (1997). A number of relatedbone morphogenetic proteins have been identified, all members of thebone morphogenetic protein (BMP) family. Bone morphogenetic native andmutant proteins, nucleic acids encoding them, related compoundsincluding receptors, host cells, and uses are further described in atleast: U.S. Pat. Nos. 5,670,338; 5,454,419; 5,661,007; 5,637,480;5,631,142; 5,166,058; 5,620,867; 5,543,394; 4,877,864; 5,013,649;5,106,748; and 5,399,677. Of particular interest are proteins havinghomology with bone morphogenetic protein 1, a procollagen C-proteinasethat plays key roles in regulating matrix deposition.

In view of the role of vascular endothelial cell growth and angiogenesisin many diseases and disorders, it is desirable to have a means ofreducing or inhibiting one or more of the biological effects causingthese processes. It is also desirable to have a means of assaying forthe presence of pathogenic polypeptides in normal and diseasedconditions, and especially cancer. Further, in a specific aspect, asthere is no generally applicable therapy for the treatment of cardiachypertrophy, the identification of factors that can prevent or reducecardiac myocyte hypertrophy is of primary importance in the developmentof new therapeutic strategies to inhibit pathophysiological cardiacgrowth. While there are several treatment modalities for variouscardiovascular and oncologic disorders, there is still a need foradditional therapeutic approaches.

The present invention is predicated upon research intended to identifynovel polypeptides which are related to VEGF and the BMP family, and inparticular, polypeptides which have a role in the survival,proliferation, and/or differentiation of cells. While the novelpolypeptides are not expected to have biological activity identical tothe known polypeptides to which they have homology, the knownpolypeptide biological activities can be used to determine the relativebiological activities of the novel polypeptides. In particular, thenovel polypeptides described herein can be used in assays which areintended to determine the ability of a polypeptide to induce survival,proliferation, or differentiation of cells. In turn, the results ofthese assays can be used accordingly, for diagnostic and therapeuticpurposes. The results of such research are the subject of the presentinvention.

SUMMARY OF THE INVENTION

Accordingly, in one aspect of the invention is provided isolated nucleicacid comprising a nucleotide sequence encoding a vascular endothelialcell growth factor-E (VEGF-E) polypeptide comprising amino acid residues1 through 345 of FIG. 2 (SEQ ID NO:2). In preferred embodiments, thisnucleic acid comprises the coding nucleotide sequence of FIG. 1 (i.e.,it comprises residues 259 through 1293 of SEQ ID NO: 1), or itscomplement. In other aspects, the invention provides a vector comprisingthis nucleic acid, preferably one that is operably linked to controlsequences recognized by a host cell transformed with the vector, as wellas a host cell comprising the nucleic acid, preferably a host celltransformed with the vector. Preferably, this host cell is a ChineseHamster Ovary cell, an insect cell, an E. coli cell, or a yeast cell,and is most preferably a baculovirus-infected insect cell.

In another embodiment, this invention provides a process for producing aVEGF-E polypeptide comprising culturing the host cell described aboveunder conditions suitable for expression of the VEGF-E polypeptide andrecovering the VEGF-E polypeptide from the cell culture. Furtherprovided is a polypeptide produced by this process.

In another embodiment, the invention provides a polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO:2.

In a further embodiment, the invention provides a chimeric polypeptidecomprising the VEGF-E polypeptide fused to a heterologous amino acidsequence. In preferred embodiments, the heterologous amino acid sequenceis an epitope tag sequence or a Fc region of an immunoglobulin.

In another aspect of the invention is provided a composition comprisingthe VEGF-E polypeptide in admixture with a carrier. In a preferredaspect, the composition comprises a therapeutically effective amount ofthe polypeptide, wherein the carrier is a pharmaceutically acceptablecarrier. Also preferred is where the composition further comprises acardiovascular, endothelial, or angiogenic agent.

In a still further embodiment, the invention provides a method forpreparing the composition for the treatment of a cardiovascular orendothelial disorder comprising admixing a therapeutically effectiveamount of the VEGF-E polypeptide with the carrier.

In another embodiment, the invention provides a pharmaceutical productcomprising:

(a) the composition described above;

(b) a container containing said composition; and

(c) a label affixed to said container, or a package insert included insaid pharmaceutical product referring to the use of said VEGF-Epolypeptide in the treatment of a cardiovascular or endothelialdisorder.

In yet another embodiment, the invention provides a method fordiagnosing a disease or a susceptibility to a disease related to amutation in a nucleic acid sequence encoding VEGF-E comprising:

(a) isolating a nucleic acid sequence encoding VEGF-E from a samplederived from a host; and

(b) determining a mutation in the nucleic acid sequence encoding VEGF-E.

In a still further embodiment, the invention provides a method ofdiagnosing cardiovascular and endothelial disorders in a mammalcomprising detecting the level of expression of a gene encoding a VEGF-Epolypeptide (a) in a test sample of tissue cells obtained from themammal, and (b) in a control sample of known normal tissue cells of thesame cell type, wherein a higher or lower expression level in the testsample indicates the presence of a cardiovascular or endothelialdysfunction in the mammal from which the test tissue cells wereobtained.

In a further embodiment, the invention provides a method for treating acardiovascular or endothelial disorder in a mammal comprisingadministering to the mammal an effective amount of a VEGF-E polypeptide.Preferably, the disorder is cardiac hypertrophy, trauma, or abone-related disorder. Also, preferably said mammal is human. In anotherpreferred embodiment, the disorder is cardiac hypertrophy and it ischaracterized by the presence of an elevated level of PGF₂, or it hasbeen induced by myocardial infarction, where preferably said VEGF-Epolypeptide administration is initiated within 48 hours followingmyocardial infarction. In another preferred embodiment, thecardiovascular or endothelial disorder is cardiac hypertrophy and saidVEGF-E polypeptide is administered together with a cardiovascular orendothelial agent. More preferably, said cardiovascular, endothelial, orangiogenic agent is selected from the group consisting of anantihypertensive drug, an ACE-inhibitor, an endothelin receptorantagonist, and a thrombolytic agent.

In another embodiment, the invention provides a method for identifyingan agonist to a VEGF-E polypeptide comprising:

(a) contacting cells and a candidate compound under conditions thatallow the polypeptide to stimulate proliferation of the cells; and

(b) measuring the extent to which cell proliferation is inhibited by thecompound.

Further provided is an agonist to a VEGF-E polypeptide identified by theabove method.

Also provided is a method for identifying a compound that inhibits theexpression or activity of a VEGF-E polypeptide, comprising:

(a) contacting a candidate compound with the polypeptide underconditions and for a time sufficient to allow the compound andpolypeptide to interact; and

(b) measuring the extent to which the compound interacts with thepolypeptide.

In another embodiment, the invention provides a compound identified bythe above method.

In a still further embodiment, the invention provides a compound thatinhibits the expression or activity of a VEGF-E polypeptide.

In another embodiment, the invention provides a method for treating anangiogenic disorder in a mammal comprising administering to the mammalan effective amount of an antagonist to a VEGF-E polypeptide. In apreferred embodiment, the angiogenic disorder is cancer or age-relatedmacular degeneration. In another preferred embodiment, the mammal ishuman. In a further preferred aspect, an effective amount of anangiostatic agent is administered in conjunction with the antagonist.

In other aspects, the invention provides an isolated antibody that bindsa VEGF-E polypeptide. Preferably, this antibody is a monoclonalantibody.

In a further aspect, the invention provides a method for inhibitingangiogenesis induced by VEGF-E polypeptide in a mammal comprisingadministering a therapeutically effective amount of the antibody to themammal, where preferably the mammal is a human. Also, the mammalpreferably has a tumor or a retinal disorder. In another preferredaspect, the mammal has cancer and the antibody is administered incombination with a chemotherapeutic agent, a growth inhibitory agent, ora cytotoxic agent.

In another preferred embodiment, the invention provides a method fordetermining the presence of a VEGF-E polypeptide comprising exposing acell suspected of containing the VEGF-E polypeptide to the antibody anddetermining binding of said antibody to said cell.

In yet another preferred aspect, the invention supplies a method ofdiagnosing cardiovascular, endothelial, or angiogenic disorders in amammal comprising (a) contacting the antibody with a test sample oftissue cells obtained from the mammal, and (b) detecting the formationof a complex between the anti-VEGF-E antibody and the VEGF-E polypeptidein the test sample.

In still further aspects, the invention provides a cancer diagnostic kitcomprising the antibody and a carrier in suitable packaging. Preferably,the kit further comprises instructions for using said antibody to detectthe VEGF-E polypeptide.

In yet another embodiment, the invention provides an article ofmanufacture, comprising:

a container;

a label on the container; and

a composition comprising an anti-VEGF-E antibody contained within thecontainer; wherein the label on the container indicates that thecomposition can be used in therapeutic or diagnostic methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a full-length DNA sequence of VEGF-E (SEQ ID NO:1), thecoding region of which is from nucleotide residues 259 through 1293. SEQID NO:1 represents DNA:29101 deposited as DNA29101-1276 Mar. 5, 1998 atthe American Type Culture Collection, Manassas, Va. It is DNA:29101,also termed UNQ:174 herein that contains the region encoding VEGF-E. Thestart and stop codon are circled, showing the coding region beginningwith ATG and the stop codon immediately after the last codingnucleotide. The coding region, 1035 nucleic acids in length, is withinSEQ ID NO:1, at positions 259 through 1293. SEQ ID NO:1 includes thenucleic acid encoding the presumed leader signal sequence orpre-protein, and the putative mature protein.

FIG. 2 depicts the deduced amino acid sequence for VEGF-E, also hereintermed PRO:200, SEQ ID NO:2. This sequence represents the proteinencoded by the open reading frame of UNQ: 174. The correspondingmolecular weight is 39,029 D. The pI is 6.06. The NX(S/T) is 3.Potential N-glycosylation sites are at positions 25, 54, and 254. CUBdomains are at positions 52-65, 118-125 and 260-273.

FIGS. 3A-3H show the effect of no growth factor (FIG. 3A), and one ormore growth factors (VEGF, bFGF, and/or PMA) (FIGS. 11B-11H) on HUVECtube formation. FIG. 3B shows VEGF, bFGF and PMA combined, FIG. 3C showsVEGF and bFGF combined, FIG. 3D shows VEGF and PMA combined, FIG. 3Eshows bFGF and PMA combined, FIG. 3F shows VEGF alone, FIG. 3G showsbFGF alone, and FIG. 3H shows PMA alone.

FIGS. 4A and 4B show, respectively, the effect on HUVEC tube formationof VEGF-E conjugated to IgG at 1% dilution and of a buffer control (10mM HEPES/0.14M NaCl/4% mannitol, pH 6.8) at 1% dilution.

FIGS. 5A and 5B show, respectively, the effect on HUVEC tube formationof VEGF-E conjugated to poly-his at 1% dilution and of a buffer control(same as in FIG. 4B) at 1% dilution.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, “vascular endothelial cell growth factor-E,” or“VEGF-E,” refers to a mammalian growth factor as described herein,including the human amino acid sequence of FIG. 2, a sequence which hashomology to VEGF and bone morphogenetic protein 1 and which includescomplete conservation of all VEGF cysteine residues, which have beenshown to be required for biological activity of VEGF. VEGF-E expressionincludes expression in human fetal bone, thymus, and thegastrointestinal tract, as well as in fetal testis, lung, and lymphnodes, and in other tissues as shown in the examples below. Thebiological activity of native VEGF-E is shared by any analogue orvariant thereof that promotes selective growth and/or survival ofumbilical vein endothelial cells, induces proliferation of pluripotentfibroblast cells, induces immediate early gene c-fos in humanendothelial cell lines, causes myocyte hypertrophy in cardiac cells,inhibits VEGF-stimulated proliferation of adrenal cortical capillaryendothelial cells, or which possesses an immune epitope that isimmunologically cross-reactive with an antibody raised against at leastone epitope of the corresponding native VEGF-E. The human VEGF-E hereinis active on rat and mouse cells, indicating conservation acrossspecies. Moreover, the VEGF-E herein is expressed at the growth plateregion and has been shown to embrace fetal myocytes.

As used herein, “vascular endothelial cell growth factor,” or “VEGF,”refers to a mammalian growth factor as defined in U.S. Pat. No.5,332,671. The biological activity of native VEGF is shared by anyanalogue or variant thereof that promotes selective growth of vascularendothelial cells but not of bovine corneal endothelial cells, lensepithelial cells, adrenal cortex cells, BHK-21 fibroblasts, orkeratinocytes, or that possesses an immune epitope that isimmunologically cross-reactive with an antibody raised against at leastone epitope of the corresponding native VEGF.

The terms “VEGF-E polypeptide” and “VEGF-E” when used herein encompassnative-sequence VEGF-E polypeptide and VEGF-E polypeptide variants(which are further defined herein). The VEGF-E polypeptides may beisolated from a variety of sources, such as from human tissue types orfrom another source, or prepared by recombinant or synthetic methods.

A “native-sequence VEGF-E polypeptide” comprises a polypeptide havingthe same amino acid sequence as a VEGF-E polypeptide derived fromnature. Such native-sequence VEGF-E polypeptide can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native-sequence VEGF-E polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of a VEGF-E polypeptide,naturally-occurring variant forms (e.g., alternatively-spliced forms)and naturally-occurring allelic variants of a VEGF-E polypeptide. In oneembodiment of the invention, the native-sequence VEGF-E polypeptide is amature or full-length native sequence VEGF-E polypeptide comprisingamino acids 1 through 345 as depicted in FIG. 2.

“VEGF-E variant” means an active VEGF-E polypeptide as defined belowhaving at least about 80% amino acid sequence identity with the VEGF-Epolypeptide having the deduced amino acid sequence shown in FIG. 2 for afull-length native-sequence VEGF-E polypeptide. Such VEGF-E polypeptidevariants include, for instance, VEGF-E polypeptides wherein one or moreamino acid residues are added, deleted, or substituted at the N- orC-terminus of the sequence of FIG. 2 or within the sequence as well asactive fragments thereof. Ordinarily, a VEGF-E polypeptide variant willhave at least about 80% amino acid sequence identity, more preferably atleast about 90% amino acid sequence identity, and even more preferablyat least about 95% amino acid sequence identity with the amino acidsequence of FIG. 2.

“Percent (%) amino acid sequence identity” with respect to the VEGF-Eamino acid sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a VEGF-E polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

“Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the sequence shown in FIG. 1 (SEQ ID NO:1), respectively, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the VEGF-Epolypeptide natural environment will not be present. Ordinarily,however, isolated polypeptide will be prepared by at least onepurification step.

An “isolated” VEGF-E polypeptide-encoding nucleic acid molecule is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the VEGF-E polypeptide-encoding nucleic acid.An isolated VEGF-E polypeptide-encoding nucleic acid molecule is otherthan in the former setting in which it is found in nature. IsolatedVEGF-E polypeptide-encoding nucleic acid molecules therefore aredistinguished from the VEGF-E polypeptide-encoding nucleic acid moleculeas it exists in natural cells. However, an isolated VEGF-Epolypeptide-encoding nucleic acid molecule includes VEGF-Epolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express VEGF-E polypeptide where, for example, the nucleicacid molecule is in a chromosomal location different from that ofnatural cells.

The phrases “cardiovascular and endothelial disorder” and“cardiovascular and endothelial dysfunction” are used interchangeablyand refer to disorders, typically systemic, that stimulate angiogenesisand/or cardiovascularization. This includes diseases that affectvessels, as well as diseases of the vessels themselves, such as of thearteries, capillaries, veins, and/or lymphatics. Such disorders include,for example, arterial disease, such as atherosclerosis, hypertension,inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon,aneurysms, and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; and other vasculardisorders such as peripheral vascular disease, trauma such as wounds,burns, and other injured tissue, implant fixation, scarring, ischemiareperfusion injury, rheumatoid arthritis, cerebrovascular disease, renaldiseases such as acute renal failure, and osteoporosis. This would alsoinclude angina, myocardial infarctions such as acute myocardialinfarctions, cardiac hypertrophy, and heart failure such as congestiveheart failure (CHF).

The phrase “angiogenic disorder” refers to a disorder that requirestreatment with an agent that inhibits angiogenesis, e.g., an angiostaticcompound. Such disorders include, for example, types of cancer such asvascular tumors, e.g., hemangioma (capillary and cavernous), glomustumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, and tumor angiogenesis.

“Hypertrophy”, as used herein, is defined as an increase in mass of anorgan or structure independent of natural growth that does not involvetumor formation. Hypertrophy of an organ or tissue is due either to anincrease in the mass of the individual cells (true hypertrophy), or toan increase in the number of cells making up the tissue (hyperplasia),or both. Certain organs, such as the heart, lose the ability to divideshortly after birth. Accordingly, “cardiac hypertrophy” is defined as anincrease in mass of the heart, which, in adults, is characterized by anincrease in myocyte cell size and contractile protein content withoutconcomitant cell division. The character of the stress responsible forinciting the hypertrophy, (e.g., increased preload, increased afterload,loss of myocytes, as in myocardial infarction, or primary depression ofcontractility), appears to play a critical role in determining thenature of the response. The early stage of cardiac hypertrophy isusually characterized morphologically by increases in the size ofmicrofibrils and mitochondria, as well as by enlargement of mitochondriaand nuclei. At this stage, while muscle cells are larger than normal,cellular organization is largely preserved. At a more advanced stage ofcardiac hypertrophy, there are preferential increases in the size ornumber of specific organelles, such as mitochondria, and new contractileelements are added in localized areas of the cells, in an irregularmanner. Cells subjected to long-standing hypertrophy show more obviousdisruptions in cellular organization, including markedly enlarged nucleiwith highly lobulated membranes, which displace adjacent myofibrils andcause breakdown of normal Z-band registration. The phrase “cardiachypertrophy” is used to include all stages of the progression of thiscondition, characterized by various degrees of structural damage of theheart muscle, regardless of the underlying cardiac disorder. Hence, theterm also includes physiological conditions instrumental in thedevelopment of cardiac hypertrophy, such as elevated blood pressure,aortic stenosis, or myocardial infarction.

“Heart failure” refers to an abnormality of cardiac function where theheart does not pump blood at the rate needed for the requirements ofmetabolizing tissues. The heart failure can be caused by a number offactors, including ischemic, congenital, rheumatic, or idiopathic forms.

“Congestive heart failure” or “CHF” is a progressive pathologic statewhere the heart is increasingly unable to supply adequate cardiac output(the volume of blood pumped by the heart over time) to deliver theoxygenated blood to peripheral tissues. As CHF progresses, structuraland hemodynamic damages occur. While these damages have a variety ofmanifestations, one characteristic symptom is ventricular hypertrophy.CHF is a common end result of a number of various cardiac disorders.

“Myocardial infarction” generally results from atherosclerosis of thecoronary arteries, often with superimposed coronary thrombosis. It maybe divided into two major types: transmural infarcts, in whichmyocardial necrosis involves the full thickness of the ventricular wall,and subendocardial (nontransmural) infarcts, in which the necrosisinvolves the subendocardium, the intramural myocardium, or both, withoutextending all the way through the ventricular wall to the epicardium.Myocardial infarction is known to cause both a change in hemodynamiceffects and an alteration in structure in the damaged and healthy zonesof the heart. Thus, for example, myocardial infarction reduces themaximum cardiac output and the stroke volume of the heart. Alsoassociated with myocardial infarction is a stimulation of the DNAsynthesis occurring in the interstice as well as an increase in theformation of collagen in the areas of the heart not affected.

As a result of the increased stress or strain placed on the heart inprolonged hypertension due, for example, to the increased totalperipheral resistance, cardiac hypertrophy has long been associated with“hypertension”. A characteristic of the ventricle that becomeshypertrophic as a result of chronic pressure overload is an impaireddiastolic performance. Fouad et al., J. Am. Coll. Cardiol., 4: 1500-1506(1984); Smith et al., J. Am. Coll. Cardiol., 5: 869-874 (1985). Aprolonged left ventricular relaxation has been detected in earlyessential hypertension, in spite of normal or supranormal systolicfunction. Hartford et al., Hypertension, 6: 329-338 (1984). However,there is no close parallelism between blood pressure levels and cardiachypertrophy. Although improvement in left ventricular function inresponse to antihypertensive therapy has been reported in humans,patients variously treated with a diuretic (hydrochlorothiazide), aβ-blocker (propranolol), or a calcium channel blocker (diltiazem), haveshown reversal of left ventricular hypertrophy, without improvement indiastolic function. Inouye et al., Am. J. Cardiol., 53: 1583-7 (1984).

Another complex cardiac disease associated with cardiac hypertrophy is“hypertrophic cardiomyopathy”. This condition is characterized by agreat diversity of morphologic, functional, and clinical features (Maronet al., N. Engl. J. Med., 316: 780-789 (1987); Spirito et al., N. Engl.J. Med., 320: 749-755 (1989); Louie and Edwards, Prog. Cardiovasc. Dis.,36: 275-308 (1994); Wigle et al., Circulation, 92: 1680-1692 (1995)),the heterogeneity of which is accentuated by the fact that it afflictspatients of all ages. Spirito et al., N. Engl. J. Med., 336: 775-785(1997). The causative factors of hypertrophic cardiomyopathy are alsodiverse and little understood. In general, mutations in genes encodingsarcomeric proteins are associated with hypertrophic cardiomyopathy.Recent data suggest that β-myosin heavy chain mutations may account forapproximately 30 to 40 percent of cases of familial hypertrophiccardiomyopathy. Watkins et al., N. Engl. J. Med., 326: 1108-1114 (1992);Schwartz et al, Circulation, 91: 532-540 (1995); Marian and Roberts,Circulation, 92: 1336-1347 (1995); Thierfelder et al., Cell, 77: 701-712(1994); Watkins et al., Nat. Gen., 11: 434-437 (1995). Besides β-myosinheavy chain, other locations of genetic mutations include cardiactroponin T, alpha topomyosin, cardiac myosin binding protein C,essential myosin light chain, and regulatory myosin light chain. SeeMalik and Watkins, Curr. Opin. Cardiol., 12: 295-302 (1997).

Supravalvular “aortic stenosis” is an inherited vascular disordercharacterized by narrowing of the ascending aorta, but other arteries,including the pulmonary arteries, may also be affected. Untreated aorticstenosis may lead to increased intracardiac pressure resulting inmyocardial hypertrophy and eventually heart failure and death. Thepathogenesis of this disorder is not fully understood, but hypertrophyand possibly hyperplasia of medial smooth muscle are prominent featuresof this disorder. It has been reported that molecular variants of theelastin gene are involved in the development and pathogenesis of aorticstenosis. U.S. Pat. No. 5,650,282 issued Jul. 22, 1997.

“Valvular regurgitation” occurs as a result of heart diseases resultingin disorders of the cardiac valves. Various diseases, like rheumaticfever, can cause the shrinking or pulling apart of the valve orifice,while other diseases may result in endocarditis, an inflammation of theendocardium or lining membrane of the atrioventricular orifices andoperation of the heart. Defects such as the narrowing of the valvestenosis or the defective closing of the valve result in an accumulationof blood in the heart cavity or regurgitation of blood past the valve.If uncorrected, prolonged valvular stenosis or insufficiency may resultin cardiac hypertrophy and associated damage to the heart muscle, whichmay eventually necessitate valve replacement.

The treatment of all these, and other cardiovascular and endothelialdisorders, which may or may not be accompanied by cardiac hypertrophy,is encompassed by the present invention.

The terms “cancer”, “cancerous”, and “malignant” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include but are notlimited to, carcinoma including adenocarcinoma, lymphoma, blastoma,melanoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer such as hepatic carcinoma and hepatoma, bladdercancer, breast cancer, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer such as renal cellcarcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, and various types of head and neck cancer. The preferred cancersfor treatment herein are breast, colon, lung, melanoma, ovarian, andothers involving vascular tumors as noted above.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., ¹³¹I,¹²⁵I, ⁹⁰Y, and ¹⁸⁶Re), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents, folic acid antagonists, anti-metabolites of nucleicacid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil,cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides,or corticosteroids. Specific examples include Adriamycin, Doxorubicin,5-Fluorouracil, Cytosine arabinoside (“Ara-C”), Cyclophosphamide,Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin,Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan, and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors, such astamoxifen and onapristone.

A “growth-inhibitory agent” when used herein refers to a compound orcomposition that inhibits growth of a cell, such as anWnt-overexpressing cancer cell, either in vitro or in vivo. Thus, thegrowth-inhibitory agent is one which significantly reduces thepercentage of malignant cells in S phase. Examples of growth-inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxol, and topo II inhibitors such as doxorubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in The Molecular Basis of Cancer, Mendelsohn and Israel, eds.,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W B Saunders: Philadelphia,1995), especially p. 13. Additional examples include tumor necrosisfactor (TNF), an antibody capable of inhibiting or neutralizing theangiogenic activity of acidic or basic FGF or hepatocyte growth factor(HGF), an antibody capable of inhibiting or neutralizing the coagulantactivities of tissue factor, protein C, or protein S (see WO 91/01753,published 21 Feb. 1991), or an antibody capable of binding to HER2receptor (WO 89/06692), such as the 4D5 antibody (and functionalequivalents thereof) (e.g., WO 92/22653).

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of acardiovascular, endothelial, or angiogenic disorder. The concept oftreatment is used in the broadest sense, and specifically includes theprevention (prophylaxis), moderation, reduction, and curing ofcardiovascular, endothelial, or angiogenic disorders of any stage.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) a cardiovascular or endothelial disorder, such ashypertrophy, or an angiogenic disorder, such as cancer. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.The disorder may result from any cause, including idiopathic,cardiotrophic, or myotrophic causes, or ischemia or ischemic insults,such as myocardial infarction.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial effect, such as an anti-hypertrophic effect, for an extendedperiod of time.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, pigs, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The phrase “cardiovascular or endothelial agents” refers generically toany drug that acts in treating cardiovascular and/or endothelialdisorders. Examples of cardiovascular agents are those that promotevascular homeostasis by modulating blood pressure, heart rate, heartcontractility, and endothelial and smooth muscle biology, all of whichfactors have a role in cardiovascular disease. Specific examples ofthese include angiotensin-II receptor antagonists; endothelin receptorantagonists such as, for example, BOSENTAN™ and MOXONODIN™;interferon-gamma (IFN-γ); des-aspartate-angiotensin I; thrombolyticagents, e.g., streptokinase, urokinase, t-PA, and a t-PA variantspecifically designed to have longer half-life and very high fibrinspecificity, TNK t-PA (a T103N, N117Q, KHRR(296-299)AAAA t-PA variant,Keyt et al., Proc. Natl. Acad. Sci. USA 91 3670-3674 (1994)); inotropicor hypertensive agents such as digoxigenin and β-adrenergic receptorblocking agents, e.g., propranolol, timolol, tertalolol, carteolol,nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, andcarvedilol; angiotensin converting enzyme (ACE) inhibitors, e.g.,quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, andlisinopril; diuretics, e.g., chorothiazide, hydrochlorothiazide,hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide,acetazolamide, and indapamide; and calcium channel blockers, e.g.,diltiazem, nifedipine, verapamil, nicardipine. One preferred category ofthis type is a therapeutic agent used for the treatment of cardiachypertrophy or of a physiological condition instrumental in thedevelopment of cardiac hypertrophy, such as elevated blood pressure,aortic stenosis, or myocardial infarction.

“Angiogenic agents” and “endothelial agents” are active agents thatpromote angiogenesis and endothelial cell growth, respectively, or, ifapplicable, vasculogenesis. This would include factors that acceleratewound healing, such as growth hormone, insulin-like growth factor-I(IGF-I), VEGF, VIGF, PDGF, epidermal growth factor (EGF), CTGF andmembers of its family, FGF, and TGF-α and TGF-β.

“Angiostatic agents” are active agents that inhibit angiogenesis orvasculogenesis or otherwise inhibit or prevent growth of cancer cells.Examples include antibodies or other antagonists to angiogenic agents asdefined above, such as antibodies to VEGF. They additionally includecytotherapeutic agents such as cytotoxic agents, chemotherapeuticagents, growth-inhibitory agents, apoptotic agents, and other agents totreat cancer, such as anti-HER-2, anti-CD20, and other bioactive andorganic chemical agents.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of an active agent (VEGF-Epolypeptide or antagonist thereto) refers to an amount effective in thetreatment of a cardiovascular, endothelial, and angiogenic disorder.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes one ormore of the biological activities of a native VEGF-E polypeptidedisclosed herein, for example, if applicable, its mitogenic orangiogenic activity. Antagonists of VEGF-E polypeptide may act byinterfering with the binding of the VEGF-E polypeptide to a cellularreceptor, by incapacitating or killing cells that have been activated byVEGF-E polypeptide, or by interfering with vascular endothelial cellactivation after VEGF-E polypeptide binding to a cellular receptor. Allsuch points of intervention by a VEGF-E polypeptide antagonist shall beconsidered equivalent for purposes of this invention. The antagonistsinhibit the mitogenic, angiogenic, or other biological activity ofVEGF-E polypeptide, and thus are useful for the treatment of diseases ordisorders characterized by undesirable excessive neovascularization,including by way of example tumors, and especially solid malignanttumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic andother retinopathies, retrolental fibroplasia, age-related maculardegeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias(including Grave's disease), corneal and other tissue transplantation,and chronic inflammation. The antagonists also are useful for thetreatment of diseases or disorders characterized by undesirableexcessive vascular permeability, such as edema associated with braintumors, ascites associated with malignancies, Meigs' syndrome, lunginflammation, nephrotic syndrome, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

In a similar manner, the term “agonist” is used in the broadest senseand includes any molecule that mimics a biological activity of a nativeVEGF-E polypeptide disclosed herein. Suitable agonist or antagonistmolecules specifically include agonist or antagonist antibodies orantibody fragments, fragments, or amino acid sequence variants of nativeVEGF-E polypeptides, peptides, small organic molecules, etc.

A “small molecule” is defined herein to have a molecular weight belowabout 500 daltons.

The term “VEGF-E polypeptide receptor” as used herein refers to acellular receptor for VEGF-E polypeptide, ordinarily a cell-surfacereceptor found on vascular endothelial cells, as well as variantsthereof that retain the ability to bind VEGF-E polypeptide.

The term “antibody” is used in the broadest sense and specificallycovers single anti-VEGF-E polypeptide monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies) and anti-VEGF-Eantibody compositions with polyepitopic specificity. The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Active” or “activity” for the purposes herein refers to form(s) ofVEGF-E which retain the biologic activities of native ornaturally-occurring VEGF-E polypeptide.

Hybridization is preferably performed under “stringent conditions” whichmeans (1) employing low ionic strength and high temperature for washing,for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C., or (2) employing during hybridization adenaturing agent, such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1%SDS. Yet another example is hybridization using a buffer of 10% dextransulfate, 2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C. Other conditions previously described and well known canbe used to arrive at high, low or moderate stringencies. When a nucleicacid sequence of a nucleic acid molecule is provided, other nucleic acidmolecules hybridizing thereto under the conditions described above areconsidered within the scope of the sequence. Preferably, the nucleicacid sequence of a nucleic acid molecule as provided herein has 70% or80% nucleic acid sequence identity to SEQ ID NO:1, positions 259 through1293. Most preferably, the nucleic acid sequence has 90% or 95% nucleicacid identity to SEQ ID NO:1, positions 259 through 1293.

“Transfection” refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

“Transformation” means introducing nucleic acid into an organism so thatthe nucleic acid is replicable, either as an extrachromosomal element orby chromosomal integrant. Depending on the host cell used,transformation is done using standard techniques appropriate to suchcells. The calcium treatment employing calcium chloride, as described byCohen, Proc. Natl. Acad. Sci. (ISA), 69: 2110 (1972) and Mandel et al.,J. Mol. Biol. 53: 154 (1970), is generally used for prokaryotes or othercells that contain substantial cell-wall barriers. For mammalian cellswithout such cell walls, the calcium phosphate precipitation method ofGraham and van der Eb, Virology, 52: 456-457 (1978) is preferred.General aspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). However, othermethods for introducing nucleic acid into cells such as by nuclearinjection or by protoplast fusion may also be used.

“Site-directed mutagenesis” is a technique standard in the art, and isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage nucleic acid to be mutagenized except for limitedmismatching, representing the desired mutation. Briefly, the syntheticoligonucleotide is used as a primer to direct synthesis of a strandcomplementary to the phage, and the resulting double-stranded nucleicacid is transformed into a phage-supporting host bacterium. Cultures ofthe transformed bacteria are plated in top agar, permitting plaqueformation from single cells that harbor the phage. Theoretically, 50% ofthe new plaques will contain the phage having, as a single strand, themutated form; 50% will have the original sequence. The plaques arehybridized with kinased synthetic primer at a temperature that permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques thathybridize with the probe are then selected and cultured, and the nucleicacid is recovered.

“Operably linked” refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence “operablylinked” to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequencesand wherein the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Forexample, nucleic acid for a presequence or secretory leader is operablylinked to nucleic acid for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, then syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

“Control sequences” refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood sequences. Eukaryoticcells are known to utilize promoters, polyadenylation signals, andenhancers.

“Expression system” refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. To effect transformation, the expression system may beincluded on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein, “cell,” “cell line,” and “cell culture” are usedinterchangeably and all such designations include progeny. Thus,“transformants” or “transformed cells” includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny that have the same functionality as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the site for which each is specific iscalled a restriction site. The various restriction enzymes used hereinare commercially available and their reaction conditions, cofactors, andother requirements as established by the enzyme suppliers are used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1mg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 ml of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein is removed by extraction with phenol and chloroform,and the digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5′ phosphates to prevent the two restriction-cleavedends of a DNA fragment from “circularizing” or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5′-terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (Maniatis et al., Molecular Cloning:A Laboratory Manual (New York: Cold Spring Harbor Laboratory, 1982), pp.133-134).

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-6114 (1981), and Goeddel et al., Nucleic Acids Res., 8, 4057(1980).

“Southern Analysis” is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. For the purposesherein, unless otherwise provided, Southern analysis shall meanseparation of digests on 1 percent agarose, denaturation, and transferto nitrocellulose by the method of Southern, J. Mol. Biol., 98: 503-517(1975), and hybridization as described by Maniatis et al., Cell, 15:687-701 (1978).

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double-stranded nucleic acid fragments (Maniatis et al., 1982,supra, p. 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase(“ligase”) per 0.5 mg of approximately equimolar amounts of the DNAfragments to be ligated.

“Preparation” of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al. 1982, supra, p. 90, may be used.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP Pat. Pub. No. 266,032published May 4, 1988, or via deoxynucleoside H-phosphonateintermediates as described by Froehler et al., Nucl. Acids Res., 14:5399-5407 (1986). They are then purified on polyacrylamide gels.

Inhibitors of VEGF-E include those which reduce or inhibit the activityor expression of VEGF-E and includes antisense molecules.

The abbreviation “KDR” refers to the kinase domain region of the VEGFmolecule. VEGF-E has no homology with VEGF in this domain.

The abbreviation “FLT-1” refers to the FMS-like tyrosine kinase bindingdomain which is known to bind to the corresponding FLT-1 receptor.VEGF-E has no homology with VEGF in this domain.

II. Compositions and Methods of the Invention

A. Full-Length VEGF-E Polypeptide

The present invention provides newly-identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas VEGF-E. In particular, cDNA encoding a VEGF-E polypeptide has beenidentified and isolated, as disclosed in further detail in the Examplesbelow. Using BLAST sequence alignment computer programs, the VEGF-Epolypeptide was found to have certain sequence identity with VEGF andBMP1.

B. VEGF-E Variants

In addition to the full-length native-sequence VEGF-E polypeptidedescribed herein, it is contemplated that VEGF-E variants can beprepared. VEGF-E variants can be prepared by introducing appropriatenucleotide changes into the VEGF-E-encoding DNA, or by synthesis of thedesired VEGF-E polypeptide. Those skilled in the art will appreciatethat amino acid changes may alter post-translational processes of theVEGF-E polypeptide, such as changing the number or position ofglycosylation sites or altering the membrane-anchoring characteristics.

Variations in the native full-length sequence VEGF-E or in variousdomains of the VEGF-E polypeptide described herein, can be made, forexample, using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion, or insertion ofone or more codons encoding the VEGF-E polypeptide that results in achange in the amino acid sequence of the VEGF-E polypeptide as comparedwith the native-sequence VEGF-E. Optionally the variation is bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the VEGF-E polypeptide. Guidance indetermining which amino acid residue may be inserted, substituted, ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the VEGF-E polypeptide with that of homologousknown protein molecules and minimizing the number of amino acid sequencechanges made in regions of high homology. Amino acid substitutions canbe the result of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, such as the replacementof a leucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of 1 to 5 aminoacids. The variation allowed may be determined by systematically makinginsertions, deletions, or substitutions of amino acids in the sequenceand testing the resulting variants for activity in the in vitro assaysdescribed in the Examples below.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)),restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)), or other known techniques can be performedon the cloned DNA to produce the VEGF-E-encoding variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions (Creighton,The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)). If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

C. Modifications of VEGF-E

Covalent modifications of VEGF-E polypeptides are included within thescope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a VEGF-E polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of a VEGF-E polypeptide.Derivatization with bifunctional agents is useful, for instance, forcrosslinking VEGF-E to a water-insoluble support matrix or surface foruse in the method for purifying anti-VEGF-E antibodies, and vice-versa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane, and agents such asmethyl-3-((p-azidophenyl)dithio)propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the VEGF-E polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native-sequence VEGF-Epolypeptide, and/or adding one or more glycosylation sites that are notpresent in the native-sequence VEGF-E polypeptide.

Addition of glycosylation sites to VEGF-E polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native-sequence VEGF-Epolypeptide (for O-linked glycosylation sites). The VEGF-E amino acidsequence may optionally be altered through changes at the DNA level,particularly by mutating the DNA encoding the VEGF-E polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theVEGF-E polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the VEGF-E polypeptide maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of VEGF-E comprises linking theVEGF-E polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

VEGF-E polypeptides of the present invention may also be modified in away to form chimeric molecules comprising a VEGF-E polypeptide fused toanother, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of a VEGF-Epolypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the VEGF-E polypeptide. Thepresence of such epitope-tagged forms of a VEGF-E polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the VEGF-E polypeptide to be readily purifiedby affinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of a VEGF-Epolypeptide with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990)). Other tag polypeptides include the Flag-peptide (Hopp et al.,BioTechnology, 6:1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science, 255:192-194 (1992)); an α-tubulin epitope peptide (Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)); and the T7 gene 10protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)).

D. Preparation of VEGF-E

The description below relates primarily to production of VEGF-E byculturing cells transformed or transfected with a vector containing atleast the coding nucleic acid shown in FIG. 1, beginning with thecircled start codon and ending just prior to the stop codon. It is, ofcourse, contemplated that alternative methods, which are well known inthe art, may be employed to prepare VEGF-E polypeptides. For instance,the VEGF-E sequence, or portions thereof, may be produced by directpeptide synthesis using solid-phase techniques (see, e.g., Stewart etal., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco,Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)). Invitro protein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of VEGF-E polypeptides maybe chemically synthesized separately and combined using chemical orenzymatic methods to produce a full-length VEGF-E polypeptide.

1. Isolation of DNA Encoding VEGF-E

DNA encoding a VEGF-E polypeptide may be obtained from a cDNA libraryprepared from tissue believed to possess the VEGF-E mRNA and to expressit at a detectable level. Accordingly, human VEGF-E-encoding DNA can beconveniently obtained from a cDNA library prepared from human tissue,such as described in the Examples. The VEGF-E-encoding gene may also beobtained from a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to a VEGF-Epolypeptide or oligonucleotides of at least about 17-80 bases) designedto identify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding VEGF-E is to use PCR methodology (Sambrook et al., supra;Dieffenbach et al., PCR Primer:A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)).

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P labeled ATP, biotinylation, or enzyme labeling. Hybridizationconditions, including low stringency, moderate stringency, and highstringency, are provided in Sambrook et al., 1989, supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as ALIGN, DNAstar, and INHERIT.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., 1989, supra, to detect precursors and processing intermediates ofmRNA that may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for VEGF-E polypeptide production and culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH, and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., 1989, supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., 1989, supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. For mammalian cells without such cell walls, thecalcium phosphate precipitation method of Graham and van der Eb,Virology, 52:456-457 (1978) can be employed. General aspects ofmammalian cell host system transformations have been described in U.S.Pat. No. 4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene or polyomithine, may also beused. For various techniques for transforming mammalian cells, see Keownet al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forVEGF-E-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism.

Suitable host cells for the expression of glycosylated VEGF-E arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the desired VEGF-Epolypeptide may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The desired VEGF-E polypeptide may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the VEGF-E-encoding DNA that is insertedinto the vector. The signal sequence may be a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV, or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theVEGF-E-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10: 157 (1980)). The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1(Jones, Genetics 85:12 (1977)).

Expression and cloning vectors usually contain a promoter operablylinked to the VEGF-E-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems (Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)).Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theVEGF-E polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

VEGF-E transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus, and SimianVirus 40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding a VEGF-E polypeptide by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theVEGF-E coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding VEGF-E.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of VEGF-E polypeptides in recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native-sequenceVEGF-E polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused toVEGF-E-encoding DNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of VEGF-E may be recovered from culture medium or from host celllysates. Cells employed in expression of VEGF-E polypeptides can bedisrupted by various physical or chemical means, such as freeze-thawcycling, sonication, mechanical disruption, or cell lysing agents. Itmay be desired to purify VEGF-E from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse-phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal-chelating columns to bind epitope-tagged forms of theVEGF-E polypeptide. Various methods of protein purification may beemployed and such methods are known in the art and described, forexample, in Deutscher, Methods in Enzymology, 182 (1990); Scopes,Protein Purification:Principles and Practice, Springer-Verlag, New York(1982). The purification step(s) selected will depend, for example, onthe nature of the production process used and the particular VEGF-Epolypeptide produced.

Since VEGF-E may aggregate into dimers, it is within the scope hereof toprovide hetero- and homodimers. Where one or more subunits are variants,the changes in amino acid sequence can be the same or different for eachsubunit chain. Heterodimers are readily produced by cotransforming hostcells with DNA encoding both subunits and, if necessary, purifying thedesired heterodimer, or by separately synthesizing the subunits,dissociating the subunits (e.g., by treatment with a chaotropic agentsuch as urea, guanidine hydrochloride, or the like), mixing thedissociated subunits, and then reassociating the subunits by dialyzingaway the chaotropic agent.

E. Uses for VEGF-E and Formulations

1. Assays for Cardiovascular, Endothelial, and Angiogenic Activity

Various assays can be used to test the polypeptide herein forcardiovascular, endothelial, and angiogenic activity. Such assaysinclude those provided in the Examples below.

Assays for testing for endothelin antagonist activity, as disclosed inU.S. Pat. No. 5,773,414, include a rat heart ventricle binding assaywhere the polypeptide is tested for its ability to inhibit iodinizedendothelin-1 binding in a receptor assay, an endothelin receptor bindingassay testing for intact cell binding of radiolabeled endothelin-1 usingrabbit renal artery vascular smooth muscle cells, an inositol phosphateaccumulation assay where functional activity is determined in Rat-1cells by measuring intra-cellular levels of second messengers, anarachidonic acid release assay that measures the ability of addedcompounds to reduce endothelin-stimulated arachidonic acid release incultured vascular smooth muscles, in vitro (isolated vessel) studiesusing endothelium from male New Zealand rabbits, and in vivo studiesusing male Sprague-Dawley rats. Assays for tissue generation activityinclude, without limitation, those described in WO 95/16035 (bone,cartilage, tendon); WO 95/05846 (nerve, neuronal), and WO 91/07491(skin, endothelium).

Assays for wound-healing activity include, for example, those describedin Winter, Epidermal Wound Healing, Maibach, H I and Rovee, D T, eds.(Year Book Medical Publishers, Inc., Chicago), pp. 71-112, as modifiedby the article of Eaglstein and Mertz, J. Invest. Dermatol., 71: 382-384(1978).

An assay to screen for a test molecule relating to a VEGF-E polypeptidethat binds an endothelin B₁ (ETB₁) receptor polypeptide and modulatessignal transduction activity involves providing a host cell transformedwith a DNA encoding endothelin B₁ receptor polypeptide, exposing thecells to the test candidate, and measuring endothelin B₁ receptor signaltransduction activity, as described, e.g., in U.S. Pat. No. 5,773,223.

There are several cardiac hypertrophy assays. In vitro assays includeinduction of spreading of adult rat cardiac myocytes. In this assay,ventricular myocytes are isolated from a single (male Sprague-Dawley)rat, essentially following a modification of the procedure described indetail by Piper et al., “Adult ventricular rat heart muscle cells” inCell Culture Techniques in Heart and Vessel Research, H. M. Piper, ed.(Berlin: Springer-Verlag, 1990), pp. 36-60. This procedure permits theisolation of adult ventricular myocytes and the long-term culture ofthese cells in the rod-shaped phenotype. Phenylephrine and ProstaglandinF_(2α) (PGF_(2α)) have been shown to induce a spreading response inthese adult cells. The inhibition of myocyte spreading induced byPGF_(2α) or PGF_(2α) analogs (e.g., fluprostenol) and phenylephrine byvarious potential inhibitors of cardiac hypertrophy is then tested.

One example of an in vivo assay is a test for inhibiting cardiachypertrophy induced by fluprostenol in vivo. This pharmacological modeltests the ability of the VEGF-E polypeptide to inhibit cardiachypertrophy induced in rats (e.g., male Wistar or Sprague-Dawley) bysubcutaneous injection of fluprostenol (an agonist analog of PGF_(2α)).It is known that rats with pathologic cardiac hypertrophy induced bymyocardial infarction have chronically elevated levels of extractablePGF_(2α) in their myocardium. Lai et al., Am. J. Physiol. (Heart Circ.Physiol.), 271: H2197-H2208 (1996). Accordingly, factors that caninhibit the effects of fluprostenol on myocardial growth in vivo arepotentially useful for treating cardiac hypertrophy. The effects of theVEGF-E polypeptide on cardiac hypertrophy are determined by measuringthe weight of heart, ventricles, and left ventricle (normalized by bodyweight) relative to fluprostenol-treated rats not receiving the VEGF-Epolypeptide.

Another example of an in vivo assay is the pressure-overload cardiachypertrophy assay. For in vivo testing it is common to inducepressure-overload cardiac hypertrophy by constriction of the abdominalaorta of test animals. In a typical protocol, rats (e.g., male Wistar orSprague-Dawley) are treated under anesthesia, and the abdominal aorta ofeach rat is narrowed down just below the diaphragm. Beznak M., Can. J.Biochem. Physiol., 33: 985-94 (1955). The aorta is exposed through asurgical incision, and a blunted needle is placed next to the vessel.The aorta is constricted with a ligature of silk thread around theneedle, which is immediately removed and which reduces the lumen of theaorta to the diameter of the needle. This approach is described, forexample, in Rossi et al., Am. Heart J., 124: 700-709 (1992) and O'Rourkeand Reibel, P.S.E.M.B. 200: 95-100 (1992).

In yet another in vivo assay, the effect on cardiac hypertrophyfollowing experimentally induced myocardial infarction (MI) is measured.Acute MI is induced in rats by left coronary artery ligation andconfirmed by electrocardiographic examination. A sham-operated group ofanimals is also prepared as control animals. Earlier data have shownthat cardiac hypertrophy is present in the group of animals with MI, asevidenced by an 18% increase in heart weight-to-body weight ratio. Laiet al., supra. Treatment of these animals with candidate blockers ofcardiac hypertrophy, e.g., VEGF-E polypeptide, provides valuableinformation about the therapeutic potential of the candidates tested.One further such assay test for induction of cardiac hypertrophy isdisclosed in U.S. Pat. No. 5,773,415, using Sprague-Dawley rats.

For cancer, a variety of well-known animal models can be used to furtherunderstand the role of the genes identified herein in the developmentand pathogenesis of tumors, and to test the efficacy of candidatetherapeutic agents, including antibodies and other antagonists of thenative VEGF-E polypeptides, such as small-molecule antagonists. The invivo nature of such models makes them particularly predictive ofresponses in human patients. Animal models of tumors and cancers (e.g.,breast cancer, colon cancer, prostate cancer, lung cancer, etc.) includeboth non-recombinant and recombinant (transgenic) animals.Non-recombinant animal models include, for example, rodent, e.g., murinemodels. Such models can be generated by introducing tumor cells intosyngeneic mice using standard techniques, e.g., subcutaneous injection,tail vein injection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, or orthopin implantation, e.g.,colon cancer cells implanted in colonic tissue. See, e.g., PCTpublication No. WO 97/33551, published Sep. 18, 1997.

Probably the most often used animal species in oncological studies areimmunodeficient mice and, in particular, nude mice. The observation thatthe nude mouse with thymic hypo/aplasia could successfully act as a hostfor human tumor xenografts has lead to its widespread use for thispurpose. The autosomal recessive nu gene has been introduced into a verylarge number of distinct congenic strains of nude mouse, including, forexample, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA,DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII, and SJL. Inaddition, a wide variety of other animals with inherited immunologicaldefects other than the nude mouse have been bred and used as recipientsof tumor xenografts. For further details see, e.g., The Nude Mouse inOncology Research, E. Boven and B. Winograd, eds. (CRC Press, Inc.,1991).

The cells introduced into such animals can be derived from knowntumor/cancer cell lines, such as any of the above-listed tumor celllines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cellline transfected with the neu protooncogene); ras-transfected NIH-3T3cells; Caco-2 (ATCC HTB-37); or a moderately well-differentiated gradeII human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38); or fromtumors and cancers. Samples of tumor or cancer cells can be obtainedfrom patients undergoing surgery, using standard conditions involvingfreezing and storing in liquid nitrogen. Karmali et al., Br. J. Cancer,48: 689-696 (1983). Tumor cells can be introduced into animals such asnude mice by a variety of procedures. The subcutaneous (s.c.) space inmice is very suitable for tumor implantation. Tumors can be transplanteds.c. as solid blocks, as needle biopsies by use of a trochar, or as cellsuspensions. For solid-block or trochar implantation, tumor tissuefragments of suitable size are introduced into the s.c. space. Cellsuspensions are freshly prepared from primary tumors or stable tumorcell lines, and injected subcutaneously. Tumor cells can also beinjected as subdermal implants. In this location, the inoculum isdeposited between the lower part of the dermal connective tissue and thes.c. tissue.

Animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the neu oncogene wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin et al. Proc. Nat. Acad. Sci. USA, 83:9129-9133 (1986).

Similarly, animal models of colon cancer can be generated by passagingcolon cancer cells in animals, e.g., nude mice, leading to theappearance of tumors in these animals. An orthotopic transplant model ofhuman colon cancer in nude mice has been described, for example, by Wanget al., Cancer Research, 54: 4726-4728 (1994) and Too et al., CancerResearch, 55: 681-684 (1995). This model is based on the so-called“METAMOUSE”™ sold by AntiCancer, Inc. (San Diego, Calif.).

Tumors that arise in animals can be removed and cultured in vitro. Cellsfrom the in vitro cultures can then be passaged to animals. Such tumorscan serve as targets for further testing or drug screening.Alternatively, the tumors resulting from the passage can be isolated andRNA from pre-passage cells and cells isolated after one or more roundsof passage analyzed for differential expression of genes of interest.Such passaging techniques can be performed with any known tumor orcancer cell lines.

For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemicallyinduced fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med.,146: 720 (1977)), which provide a highly controllable model system forstudying the anti-tumor activities of various agents. Palladino et al.,J. Immunol., 138: 4023-4032 (1987). Briefly, tumor cells are propagatedin vitro in cell culture. Prior to injection into the animals, the celllines are washed and suspended in buffer, at a cell density of about10×10⁶ to 10×10⁷ cells/ml. The animals are then infected subcutaneouslywith 10 to 100 μl of the cell suspension, allowing one to three weeksfor a tumor to appear.

In addition, the Lewis lung (3LL) carcinoma of mice, which is one of themost thoroughly studied experimental tumors, can be used as aninvestigational tumor model. Efficacy in this tumor model has beencorrelated with beneficial effects in the treatment of human patientsdiagnosed with small-cell carcinoma of the lung (SCCL). This tumor canbe introduced in normal mice upon injection of tumor fragments from anaffected mouse or of cells maintained in culture. Zupi et al., Br. J.Cancer, 41: suppl. 4, 30 (1980). Evidence indicates that tumors can bestarted from injection of even a single cell and that a very highproportion of infected tumor cells survive. For further informationabout this tumor model see Zacharski, Haemostasis, 16: 300-320 (1986).

One way of evaluating the efficacy of a test compound in an animal modelwith an implanted tumor is to measure the size of the tumor before andafter treatment. Traditionally, the size of implanted tumors has beenmeasured with a slide caliper in two or three dimensions. The measurelimited to two dimensions does not accurately reflect the size of thetumor; therefore, it is usually converted into the corresponding volumeby using a mathematical formula. However, the measurement of tumor sizeis very inaccurate. The therapeutic effects of a drug candidate can bebetter described as treatment-induced growth delay and specific growthdelay. Another important variable in the description of tumor growth isthe tumor volume doubling time. Computer programs for the calculationand description of tumor growth are also available, such as the programreported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop onImmune-Deficient Animals, Wu and Sheng eds. (Basel, 1989), p. 301. It isnoted, however, that necrosis and inflammatory responses followingtreatment may actually result in an increase in tumor size, at leastinitially. Therefore, these changes need to be carefully monitored, by acombination of a morphometric method and flow cytometric analysis.

Further, nucleic acids that encode VEGF-E polypeptide or any of itsmodified forms can also be used to generate either transgenic animals or“knock-out” animals which, in turn, are useful in the development andscreening of therapeutically useful reagents. A transgenic animal (e.g.,a mouse or rat) is an animal having cells that contain a transgene,which transgene was introduced into the animal or an ancestor of theanimal at a prenatal, e.g., an embryonic stage. A transgene is a DNAwhich is integrated into the genome of a cell from which a transgenicanimal develops. Hence, recombinant (transgenic) animal models can beengineered by introducing the coding portion of the genes encodingVEGF-E identified herein into the genome of animals of interest, usingstandard techniques for producing transgenic animals. Animals that canserve as a target for transgenic manipulation include, withoutlimitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, andnon-human primates, e.g., baboons, chimpanzees and monkeys. In oneembodiment, cDNA encoding VEGF-E polypeptide can be used to clonegenomic DNA encoding VEGF-E in accordance with established techniquesand the genomic sequences used to generate transgenic animals thatcontain cells which express DNA encoding VEGF-E. Techniques known in theart to introduce a transgene into such animals include pronucleicmicroinjection (U.S. Pat. No. 4,873,191); retrovirus-mediated genetransfer into germ lines (e.g., Van der Putten et al., Proc. Natl. Acad.Sci. USA, 82: 6148-615 (1985)); gene targeting in embryonic stem cells(Thompson et al., Cell 56: 313-321 (1989)); electroporation of embryos(Lo, Mol. Cell. Biol., 3: 1803-1814 (1983)); and sperm-mediated genetransfer. Lavitrano et al., Cell, 57: 717-73 (1989). For a review, see,for example, U.S. Pat. No. 4,736,866. Methods for generating transgenicanimals, particularly animals such as mice or rats, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for VEGF-E transgene incorporation with tissue-specificenhancers. Transgenic animals that include a copy of a transgeneencoding VEGF-E introduced into the germ line of the animal at anembryonic stage can be used to examine the effect of increasedexpression of DNA encoding VEGF-E. Such animals can be used as testeranimals for reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA, 89: 6232-636 (1992). The expression of the transgene intransgenic animals can be monitored by standard techniques. For example,Southern blot analysis or PCR amplification can be used to verify theintegration of the transgene. The level of mRNA expression can then beanalyzed using techniques such as in situ hybridization, Northern blotanalysis, PCR, or immunocytochemistry. The animals are further examinedfor signs of tumor or cancer development.

Alternatively, “knock-out” animals can be constructed that have adefective or altered gene encoding a VEGF-E polypeptide identifiedherein, as a result of homologous recombination between the endogenousgene encoding the VEGF-E polypeptide and altered genomic DNA encodingthe same polypeptide introduced into an embryonic cell of the animal.For example, cDNA encoding a particular VEGF-E polypeptide can be usedto clone genomic DNA encoding that polypeptide in accordance withestablished techniques. A portion of the genomic DNA encoding aparticular VEGF-E polypeptide can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector. See, e.g.,Thomas and Capecchi, Cell, 51: 503 (1987) for a description ofhomologous recombination vectors. The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced DNA has homologously recombined with the endogenous DNAare selected. See, e.g., Li et al., Cell, 69: 915 (1992). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse orrat) to form aggregation chimeras. See, e.g., Bradley, inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL: Oxford, 1987), pp. 113-152. A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term to create a “knock-out” animal. Progenyharboring the homologously recombined DNA in their germ cells can beidentified by standard techniques and used to breed animals in which allcells of the animal contain the homologously recombined DNA. Knockoutanimals can be characterized, for instance, by their ability to defendagainst certain pathological conditions and by their development ofpathological conditions due to absence of the VEGF-E polypeptide.

The efficacy of antibodies specifically binding the VEGF-E polypeptidesidentified herein, and other drug candidates, can be tested also in thetreatment of spontaneous animal tumors. A suitable target for suchstudies is the feline oral squamous cell carcinoma (SCC). Feline oralSCC is a highly invasive, malignant tumor that is the most common oralmalignancy of cats, accounting for over 60% of the oral tumors reportedin this species. It rarely metastasizes to distant sites, although thislow incidence of metastasis may merely be a reflection of the shortsurvival times for cats with this tumor. These tumors are usually notamenable to surgery, primarily because of the anatomy of the feline oralcavity. At present, there is no effective treatment for this tumor.Prior to entry into the study, each cat undergoes complete clinicalexamination and biopsy, and is scanned by computed tomography (CT). Catsdiagnosed with sublingual oral squamous cell tumors are excluded fromthe study. The tongue can become paralyzed as a result of such tumor,and even if the treatment kills the tumor, the animals may not be ableto feed themselves. Each cat is treated repeatedly, over a longer periodof time. Photographs of the tumors will be taken daily during thetreatment period, and at each subsequent recheck. After treatment, eachcat undergoes another CT scan. CT scans and thoracic radiograms areevaluated every 8 weeks thereafter. The data are evaluated fordifferences in survival, response, and toxicity as compared to controlgroups. Positive response may require evidence of tumor regression,preferably with improvement of quality of life and/or increased lifespan.

In addition, other spontaneous animal tumors, such as fibrosarcoma,adenocarcinoma, lymphoma, chondroma, or leiomyosarcoma of dogs, cats,and baboons can also be tested. Of these, mammary adenocarcinoma in dogsand cats is a preferred model as its appearance and behavior are verysimilar to those in humans. However, the use of this model is limited bythe rare occurrence of this type of tumor in animals.

Other in vitro and in vivo cardiovascular, endothelial, and angiogenictests known in the art are also suitable herein.

2. Tissue Distribution

The results of the cardiovascular, endothelial, and angiogenic assaysherein can be verified by further studies, such as by determining mRNAexpression in various human tissues.

As noted before, gene amplification and/or gene expression in varioustissues may be measured by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native-sequenceVEGF-E polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to DNAencoding VEGF-E and encoding a specific antibody epitope. Generaltechniques for generating antibodies, and special protocols for in situhybridization are provided hereinbelow.

3. Antibody Binding Studies

The results of the cardiovascular, endothelial, and angiogenic study canbe further verified by antibody binding studies, in which the ability ofanti-VEGF-E antibodies to inhibit the effect of the VEGF-E polypeptideson endothelial cells or other cells used in the cardiovascular,endothelial, and angiogenic assays is tested. Exemplary antibodiesinclude polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies, the preparation of which will be describedhereinbelow.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques (CRC Press, Inc., 1987), pp. 147-158.

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte that remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody that is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

4. Cell-Based Tumor Assays

Cell-based assays and animal models for cardiovascular, endothelial, andangiogenic disorders, such as tumors, can be used to verify the findingsof a cardiovascular, endothelial, and angiogenic assay herein, andfurther to understand the relationship between the genes identifiedherein and the development and pathogenesis of undesirablecardiovascular, endothelial, and angiogenic cell growth. The role ofgene products identified herein in the development and pathology ofundesirable cardiovascular, endothelial, and angiogenic cell growth,e.g., tumor cells, can be tested by using cells or cells lines that havebeen identified as being stimulated or inhibited by the VEGF-Epolypeptide herein. Such cells include, for example, those set forth inthe Examples below.

In a different approach, cells of a cell type known to be involved in aparticular cardiovascular, endothelial, and angiogenic disorder aretransfected with the cDNAs herein, and the ability of these cDNAs toinduce excessive growth or inhibit growth is analyzed. If thecardiovascular, endothelial, and angiogenic disorder is cancer, suitabletumor cells include, for example, stable tumor cells lines such as theB104-1-1 cell line (stable NIH-3T3 cell line transfected with the neuprotooncogene) and ras-transfected NIH-3T3 cells, which can betransfected with the desired gene and monitored for tumorigenic growth.Such transfected cell lines can then be used to test the ability ofpoly- or monoclonal antibodies or antibody compositions to inhibittumorigenic cell growth by exerting cytostatic or cytotoxic activity onthe growth of the transformed cells, or by mediating antibody-dependentcellular cytotoxicity (ADCC). Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of cardiovascular, endothelial, andangiogenic disorders such as cancer.

In addition, primary cultures derived from tumors in transgenic animals(as described above) can be used in the cell-based assays herein,although stable cell lines are preferred. Techniques to derivecontinuous cell lines from transgenic animals are well known in the art.See, e.g., Small et al., Mol. Cell. Biol. 5: 642-648 (1985).

5. Gene Therapy

The VEGF-E polypeptide herein and polypeptidyl agonists and antagonistsmay be employed in accordance with the present invention by expressionof such polypeptides in vivo, which is often referred to as genetherapy.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells: in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the sites where the VEGF-E polypeptide is required,i.e., the site of synthesis of the VEGF-E polypeptide, if known, and thesite (e.g., wound) where VEGF-E polypeptide biological activity isneeded. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells, and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes that are implanted into the patient(see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187).

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or transferredin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, transduction, cellfusion, DEAE-dextran, the calcium phosphate precipitation method, etc.Transduction involves the association of a replication-defective,recombinant viral (preferably retroviral) particle with a cellularreceptor, followed by introduction of the nucleic acids contained by theparticle into the cell. A commonly used vector for ex vivo delivery ofthe gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral vectors (such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) andlipid-based systems (useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol; see, e.g., Tonkinson etal., Cancer Investigation, 14(1): 54-65 (1996)). The most preferredvectors for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral vector such asa retroviral vector includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger. Inaddition, a viral vector such as a retroviral vector includes a nucleicacid molecule that, when transcribed in the presence of a gene encodingVEGF-E polypeptide, is operably linked thereto and acts as a translationinitiation sequence. Such vector constructs also include a packagingsignal, long terminal repeats (LTRs) or portions thereof, and positiveand negative strand primer binding sites appropriate to the virus used(if these are not already present in the viral vector). In addition,such vector typically includes a signal sequence for secretion of theVEGF-E polypeptide from a host cell in which it is placed. Preferablythe signal sequence for this purpose is a mammalian signal sequence,most preferably the native signal sequence for VEGF-E polypeptide.Optionally, the vector construct may also include a signal that directspolyadenylation, as well as one or more restriction sites and atranslation termination sequence. By way of example, such vectors willtypically include a 5′ LTR, a tRNA binding site, a packaging signal, anorigin of second-strand DNA synthesis, and a 3′ LTR or a portionthereof. Other vectors can be used that are non-viral, such as cationiclipids, polylysine, and dendrimers.

In some situations, it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell-surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins that bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g, capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins that undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For a review of the currentlyknown gene marking and gene therapy protocols, see Anderson et al.,Science, 256: 808-813 (1992). See also WO 93/25673 and the referencescited therein.

Suitable gene therapy and methods for making retroviral particles andstructural proteins can be found in, e.g., U.S. Pat. No. 5,681,746.

6. Use of Gene as Diagnostic

This invention is also related to the use of the gene encoding theVEGF-E polypeptide as a diagnostic. Detection of a mutated form of theVEGF-E polypeptide will allow a diagnosis of a cardiovascular,endothelial, and angiogenic disease or a susceptibility to acardiovascular, endothelial, and angiogenic disease, such as a tumor,since mutations in the VEGF-E polypeptide may cause tumors.

Individuals carrying mutations in the gene encoding human VEGF-Epolypeptide may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy, and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324: 163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding theVEGF-E polypeptide can be used to identify and analyze VEGF-Epolypeptide mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled RNA encoding VEGF-E polypeptide, oralternatively, radiolabeled antisense DNA sequences encoding VEGF-Epolypeptide. Perfectly matched sequences can be distinguished frommismatched duplexes by RNAse A digestion or by differences in meltingtemperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamidine gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures. See, e.g., Myerset al., Science, 230: 1242 (1985).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNAse and S1 protection or the chemicalcleavage method, for example, Cotton et al., Proc. Natl. Acad. Sci. USA,85: 4397-4401 (1985).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNAse protection, chemical cleavage,direct DNA sequencing, or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms (RFLP), and Southern blottingof genomic DNA.

7. Use to Detect VEGF-E Polypeptide Levels

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

VEGF-E polypeptide expression may be linked to vascular disease orneovascularization associated with tumor formation. If the VEGF-Epolypeptide has a signal sequence and the mRNA is highly expressed inendothelial cells and to a lesser extent in smooth muscle cells, thisindicates that the VEGF-E polypeptide is present in serum. Accordingly,an anti-VEGF-E polypeptide antibody could be used to diagnose vasculardisease or neovascularization associated with tumor formation, since analtered level of this VEGF-E polypeptide may be indicative of suchdisorders.

A competition assay may be employed wherein antibodies specific to theVEGF-E polypeptide are attached to a solid support and labeled VEGF-Epolypeptide and a sample derived from the host are passed over the solidsupport and the amount of label detected attached to the solid supportcan be correlated to a quantity of VEGF-E polypeptide in the sample.

8. Probes and Immunoassays

VEGF-E amino acid variant sequences and derivatives that areimmunologically crossreactive with antibodies raised against native VEGFare useful in immunoassays for VEGF-E as standards, or, when labeled, ascompetitive reagents.

The full-length nucleotide sequence SEQ ID NO:1, or portions thereof,may be used as hybridization probes for a cDNA library to isolate thefull-length VEGF-E gene or to isolate still other genes (for instance,those encoding naturally-occurring variants of VEGF-E or VEGF-E fromother species) which have a desired sequence identity to the VEGF-Esequence disclosed in FIG. 1 (SEQ ID NO:1). Optionally, the length ofthe probes will be about 17 to about 50 bases. The hybridization probesmay be derived from the nucleotide sequence of SEQ ID NO:1 as shown inFIG. 1 or from genomic sequences including promoters, enhancer elements,and introns of native-sequence VEGF-E-encoding DNA. By way of example, ascreening method will comprise isolating the coding region of the VEGF-Egene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the VEGF-E gene of the present invention can beused to screen libraries of human cDNA, genomic DNA, or mRNA todetermine which members of such libraries the probe hybridizes to.Hybridization techniques are described in further detail in the Examplesbelow.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related VEGF-E sequences.

9. Chromosome Mapping

Nucleotide sequences encoding a VEGF-E polypeptide can also be used toconstruct hybridization probes for mapping the gene which encodes thatVEGF-E polypeptide and for the genetic analysis of individuals withgenetic disorders. The nucleotide sequence provided herein may be mappedto a chromosome and specific regions of a chromosome using knowntechniques, such as in situ hybridization, linkage analysis againstknown chromosomal markers, and hybridization screening with libraries.

For chromosome identification, the sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease. Briefly, sequences can bemapped to chromosomes by preparing PCR primers (preferably 15-25 bp)from the cDNA. Computer analysis for the 3′ untranslated region is usedto rapidly select primers that do not span more than one exon in thegenomic DNA, thus complicating the amplification process. These primersare then used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes, andpreselection by hybridization to construct chromosome-specific cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases; however, clones larger than 2,000 bp have a higher likelihood ofbinding to a unique chromosomal location with sufficient signalintensity for simple detection. FISH requires use of the clones fromwhich the gene encoding VEGF-E polypeptide was derived, and the longerthe better. For example, 2,000 bp is good, 4,000 bp is better, and morethan 4,000 is probably not necessary to get good results a reasonablepercentage of the time. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques (Pergamon Press,New York, 1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region is thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

10. Screening Assays for Drug Candidates

Screening assays can be designed to find lead compounds that mimic thebiological activity of a native VEGF-E or a receptor for VEGF-E. Suchscreening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates. Small molecules contemplatedinclude synthetic organic or inorganic compounds. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art.

Hence, this invention encompasses methods of screening compounds toidentify those that mimic the VEGF-E polypeptide (agonists) or preventthe effect of the VEGF-E polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the VEGF-E polypeptides encoded by the genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a VEGF-E polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the VEGF-E polypeptide encoded by the gene identified hereinor the drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the VEGF-E polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for theVEGF-E polypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular VEGF-E polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340: 245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding aVEGF-E polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

If the VEGF-E polypeptide has the ability to stimulate the proliferationof endothelial cells in the presence of the co-mitogen ConA, then oneexample of a screening method takes advantage of this ability.Specifically, in the proliferation assay, human umbilical veinendothelial cells are obtained and cultured in 96-well flat-bottomedculture plates (Costar, Cambridge, Mass.) and supplemented with areaction mixture appropriate for facilitating proliferation of thecells, the mixture containing Con-A (Calbiochem, La Jolla, Calif.).Con-A and the compound to be screened are added and after incubation at37° C., cultures are pulsed with ³⁻H-thymidine and harvested onto glassfiber filters (phD; Cambridge Technology, Watertown, Mass.). Mean³⁻(H)thymidine incorporation (cpm) of triplicate cultures is determinedusing a liquid scintillation counter (Beckman Instruments, Irvine,Calif.). Significant ³⁻(H)thymidine incorporation indicates stimulationof endothelial cell proliferation.

To assay for antagonists, the assay described above is performed;however, in this assay the VEGF-E polypeptide is added along with thecompound to be screened and the ability of the compound to inhibit³⁻(H)thymidine incorporation in the presence of the VEGF-E polypeptideindicates that the compound is an antagonist to the VEGF-E polypeptide.Alternatively, antagonists may be detected by combining the VEGF-Epolypeptide and a potential antagonist with membrane-bound VEGF-Epolypeptide receptors or recombinant receptors under appropriateconditions for a competitive inhibition assay. The VEGF-E polypeptidecan be labeled, such as by radioactivity, such that the number of VEGF-Epolypeptide molecules bound to the receptor can be used to determine theeffectiveness of the potential antagonist. The gene encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting. Coligan etal., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the VEGF-E polypeptide and a cDNA librarycreated from this RNA is divided into pools and used to transfect COScells or other cells that are not responsive to the VEGF-E polypeptide.Transfected cells that are grown on glass slides are exposed to labeledVEGF-E polypeptide. The VEGF-E polypeptide can be labeled by a varietyof means including iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to autoradiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aninteractive sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor.

As an alternative approach for receptor identification, labeled VEGF-Epolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledVEGF-E polypeptide in the presence of the candidate compound. Theability of the compound to enhance or block this interaction could thenbe measured. The compositions useful in the treatment of cardiovascular,endothelial, and angiogenic disorders include, without limitation,antibodies, small organic and inorganic molecules, peptides,phosphopeptides, antisense and ribozyme molecules, triple-helixmolecules, etc., that inhibit the expression and/or activity of thetarget gene product.

More specific examples of potential antagonists include anoligonucleotide that binds to the VEGF-E polypeptide,(poly)peptide-immunoglobulin fusions, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. Alternatively, apotential antagonist may be a closely related protein, for example, amutated form of the VEGF-E polypeptide that recognizes the receptor butimparts no effect, thereby competitively inhibiting the action of theVEGF-E polypeptide.

Another potential VEGF-E polypeptide antagonist is an antisense RNA orDNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature VEGF-E polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6: 3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of the VEGF-E polypeptide. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the VEGF-E polypeptide (antisense—Okano,Neurochem., 56: 560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the VEGF-E polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the VEGF-E polypeptide, thereby blocking the normalbiological activity of the VEGF-E polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4: 469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

11. Types of Cardiovascular, Endothelial, and Angiogenic Disorders to beTreated

The VEGF-E polypeptides, or agonists or antagonists thereto, that haveactivity in the cardiovascular, angiogenic, and endothelial assaysdescribed herein, and/or whose gene product has been found to belocalized to the cardiovascular system, are likely to have therapeuticuses in a variety of cardiovascular, endothelial, and angiogenicdisorders, including systemic disorders that affect vessels, such asdiabetes mellitus. The VEGF-E molecules herein have a number oftherapeutic uses associated with survival, proliferation and/ordifferention of cells. Such uses include the treatment of umbilical veinendothelial cells, in view of the demonstrated ability of VEGF-E toincrease survival of human umbilical vein endothelial cells. Treatmentmay be needed if the vein were subjected to traumata, or situationswherein artificial means are employed to enhance the survival of theumbilical vein, for example, where it is weak, diseased, based on anartificial matrix, or in an artificial environment. Other physiologicalconditions that could be improved based on the selective mitogeniccharacter of VEGF-E are also included herein. Uses also include thetreatment of fibroblasts and myocytes, in view of the demonstratedability of VEGF-E to induce proliferation of fibroblasts and hypertrophyin myocytes. In particular, VEGF-E can be used in wound healing, tissuegrowth and muscle generation and regeneration.

Their therapeutic utility could include diseases of the arteries,capillaries, veins, and/or lymphatics. Examples of treatments hereunderinclude treating muscle wasting disease, treating osteoporosis, aidingin implant fixation to stimulate the growth of cells around the implantand therefore facilitate its attachment to its intended site, increasingIGF stability in tissues or in serum, if applicable, and increasingbinding to the IGF receptor (since IGF has been shown in vitro toenhance human marrow erythroid and granulocytic progenitor cell growth).

The VEGF-E polypeptides or agonists or antagonists thereto may also beemployed to stimulate erythropoiesis or granulopoiesis, to stimulatewound healing or tissue regeneration and associated therapies concernedwith re-growth of tissue, such as connective tissue, skin, bone,cartilage, muscle, lung, or kidney, to promote angiogenesis, tostimulate or inhibit migration of endothelial cells, and to proliferatethe growth of vascular smooth muscle and endothelial cell production.The increase in angiogenesis mediated by VEGF-E polypeptide orantagonist would be beneficial to ischemic tissues and to collateralcoronary development in the heart subsequent to coronary stenosis.Antagonists are used to inhibit the action of such polypeptides, forexample, to limit the production of excess connective tissue duringwound healing or pulmonary fibrosis if the VEGF-E polypeptide promotessuch production. This would include treatment of acute myocardialinfarction and heart failure.

Moreover, the present invention concerns the treatment of cardiachypertrophy, regardless of the underlying cause, by administering atherapeutically effective dose of VEGF-E polypeptide, or agonist orantagonist thereto. If the objective is the treatment of human patients,the VEGF-E polypeptide preferably is recombinant human VEGF-Epolypeptide (rhVEGF-E polypeptide). The treatment for cardiachypertrophy can be performed at any of its various stages, which mayresult from a variety of diverse pathologic conditions, includingmyocardial infarction, hypertension, hypertrophic cardiomyopathy, andvalvular regurgitation. The treatment extends to all stages of theprogression of cardiac hypertrophy, with or without structural damage ofthe heart muscle, regardless of the underlying cardiac disorder.

The decision of whether to use the molecule itself or an agonist thereoffor any particular indication, as opposed to an antagonist to themolecule, would depend mainly on whether the molecule herein promotescardiovascularization, genesis of endothelial cells, or angiogenesis orinhibits these conditions. For example, if the molecule promotesangiogenesis, an antagonist thereof would be useful for treatment ofdisorders where it is desired to limit or prevent angiogenesis. Examplesof such disorders include vascular tumors such as haemangioma, tumorangiogenesis, neovascularization in the retina, choroid, or cornea,associated with diabetic retinopathy or premature infant retinopathy ormacular degeneration and proliferative vitreoretinopathy, rheumatoidarthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation,psoriasis, endometriosis associated with neovascularization, restenosissubsequent to balloon angioplasty, scar tissue overproduction, forexample, that seen in a keloid that forms after surgery, fibrosis aftermyocardial infarction, or fibrotic lesions associated with pulmonaryfibrosis.

If, however, the molecule inhibits angiogenesis, it would be expected tobe used directly for treatment of the above conditions.

On the other hand, if the molecule stimulates angiogenesis it would beused itself (or an agonist thereof) for indications where angiogenesisis desired such as peripheral vascular disease, hypertension,inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon,aneurysms, arterial restenosis, thrombophlebitis, lymphangitis,lymphedema, wound healing and tissue repair, ischemia reperfusioninjury, angina, myocardial infarctions such as acute myocardialinfarctions, chronic heart conditions, heart failure such as congestiveheart failure, and osteoporosis. If, however, the molecule inhibitsangiogenesis, an antagonist thereof would be used for treatment of thoseconditions where angiogenesis is desired.

Specific types of diseases are described below, where the VEGF-Epolypeptide herein or antagonists thereof may serve as useful forvascular-related drug targeting or as therapeutic targets for thetreatment or prevention of the disorders. Atherosclerosis is a diseasecharacterized by accumulation of plaques of intimal thickening inarteries, due to accumulation of lipids, proliferation of smooth musclecells, and formation of fibrous tissue within the arterial wall. Thedisease can affect large, medium, and small arteries in any organ.Changes in endothelial and vascular smooth muscle cell function areknown to play an important role in modulating the accumulation andregression of these plaques.

Hypertension is characterized by raised vascular pressure in thesystemic arterial, pulmonary arterial, or portal venous systems.Elevated pressure may result from or result in impaired endothelialfunction and/or vascular disease.

Inflammatory vasculitides include giant cell arteritis, Takayasu'sarteritis, polyarteritis nodosa (including the microangiopathic form),Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis,and a variety of infectious-related vascular disorders (includingHenoch-Schonlein prupura). Altered endothelial cell function has beenshown to be important in these diseases.

Reynaud's disease and Reynaud's phenomenon are characterized byintermittent abnormal impairment of the circulation through theextremities on exposure to cold. Altered endothelial cell function hasbeen shown to be important in this disease.

Aneurysms are saccular or fusiform dilatations of the arterial or venoustree that are associated with altered endothelial cell and/or vascularsmooth muscle cells.

Arterial restenosis (restenosis of the arterial wall) may occurfollowing angioplasty as a result of alteration in the function andproliferation of endothelial and vascular smooth muscle cells.

Thrombophlebitis and lymphangitis are inflammatory disorders of veinsand lymphatics, respectively, that may result from, and/or in, alteredendothelial cell function. Similarly, lymphedema is a conditioninvolving impaired lymphatic vessels resulting from endothelial cellfunction.

The family of benign and malignant vascular tumors are characterized byabnormal proliferation and growth of cellular elements of the vascularsystem. For example, lymphangiomas are benign tumors of the lymphaticsystem that are congenital, often cystic, malformations of thelymphatics that usually occur in newborns. Cystic tumors tend to growinto the adjacent tissue. Cystic tumors usually occur in the cervicaland axillary region. They can also occur in the soft tissue of theextremities. The main symptoms are dilated, sometimes reticular,structured lymphatics and lymphocysts surrounded by connective tissue.Lymphangiomas are assumed to be caused by improperly connected embryoniclymphatics or their deficiency. The result is impaired local lymphdrainage. Griener et al., Lmphology, 4: 140-144 (1971).

Another use for the VEGF-E polypeptides herein or antagonists thereto isin the prevention of tumor angiogenesis, which involves vascularizationof a tumor to enable it to growth and/or metastasize. This process isdependent on the growth of new blood vessels. Examples of neoplasms andrelated conditions that involve tumor angiogenesis include breastcarcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas,colorectal carcinomas, liver carcinomas, ovarian carcinomas, the comas,arrhenoblastomas, cervical carcinomas, endometrial carcinoma,endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas,hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma,cavernous hemangioma, hemangioblastoma, pancreas carcinomas,retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma,abnormal vascular proliferation associated with phakomatoses, edema(such as that associated with brain tumors), and Meigs' syndrome.

Age-related macular degeneration (AMD) is a leading cause of severevisual loss in the elderly population. The exudative form of AMD ischaracterized by choroidal neovascularization and retinal pigmentepithelial cell detachment. Because choroidal neovascularization isassociated with a dramatic worsening in prognosis, the VEGF-Epolypeptides or antagonist thereto is expected to be useful in reducingthe severity of AMD.

Healing of trauma such as wound healing and tissue repair is also atargeted use for the VEGF-E polypeptides herein or their antagonists.Formation and regression of new blood vessels is essential for tissuehealing and repair. This category includes bone, cartilage, tendon,ligament, and/or nerve tissue growth or regeneration, as well as woundhealing and tissue repair and replacement, and in the treatment ofburns, incisions, and ulcers. A VEGF-E polypeptide or antagonist thereofthat induces cartilage and/or bone growth in circumstances where bone isnot normally formed has application in the healing of bone fractures andcartilage damage or defects in humans and other animals. Such apreparation employing a VEGF-E polypeptide or antagonist thereof mayhave prophylactic use in closed as well as open fracture reduction andalso in the improved fixation of artificial joints. De novo boneformation induced by an osteogenic agent contributes to the repair ofcongenital, trauma-induced, or oncologic, resection-induced craniofacialdefects, and also is useful in cosmetic plastic surgery.

VEGF-E polypeptides or antagonists thereto may also be useful to promotebetter or faster closure of non-healing wounds, including withoutlimitation pressure ulcers, ulcers associated with vascularinsufficiency, surgical and traumatic wounds, and the like.

It is expected that a VEGF-E polypeptide or antagonist thereto may alsoexhibit activity for generation or regeneration of other tissues, suchas organs (including, for example, pancreas, liver, intestine, kidney,skin, or endothelium), muscle (smooth, skeletal, or cardiac), andvascular (including vascular endothelium) tissue, or for promoting thegrowth of cells comprising such tissues. Part of the desired effects maybe by inhibition or modulation of fibrotic scarring to allow normaltissue to regenerate.

A VEGF-E polypeptide herein or antagonist thereto may also be useful forgut protection or regeneration and treatment of lung or liver fibrosis,reperfusion injury in various tissues, and conditions resulting fromsystemic cytokine damage. Also, the VEGF-E polypeptide or antagonistthereto may be useful for promoting or inhibiting differentiation oftissues described above from precursor tissues or cells, or forinhibiting the growth of tissues described above.

A VEGF-E polypeptide or antagonist thereto may also be used in thetreatment of periodontal diseases and in other tooth-repair processes.Such agents may provide an environment to attract bone-forming cells,stimulate growth of bone-forming cells, or induce differentiation ofprogenitors of bone-forming cells. A VEGF-E polypeptide herein or anantagonist thereto may also be useful in the treatment of osteoporosisor osteoarthritis, such as through stimulation of bone and/or cartilagerepair or by blocking inflammation or processes of tissue destruction(collagenase activity, osteoclast activity, etc.) mediated byinflammatory processes, since blood vessels play an important role inthe regulation of bone turnover and growth.

Another category of tissue regeneration activity that may beattributable to the VEGF-E polypeptide herein or antagonist thereto istendon/ligament formation. A protein that induces tendon/ligament-liketissue or other tissue formation in circumstances where such tissue isnot normally formed has application in the healing of tendon or ligamenttears, deformities, and other tendon or ligament defects in humans andother animals. Such a preparation may have prophylactic use inpreventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of theVEGF-E polypeptide herein or antagonist thereto contributes to therepair of congenital, trauma-induced, or other tendon or ligamentdefects of other origin, and is also useful in cosmetic plastic surgeryfor attachment or repair of tendons or ligaments. The compositionsherein may provide an environment to attract tendon- or ligament-formingcells, stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions herein may also beuseful in the treatment of tendinitis, carpal tunnel syndrome, and othertendon or ligament defects. The compositions may also include anappropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The VEGF-E polypeptide or its antagonist may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e., for the treatment of central and peripheral nervous systemdisease and neuropathies, as well as mechanical and traumatic disorders,that involve degeneration, death, or trauma to neural cells or nervetissue. More specifically, a VEGF-E polypeptide or its antagonist may beused in the treatment of diseases of the peripheral nervous system, suchas peripheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions that may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma, andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a VEGF-E polypeptide herein or antagonist thereto.

Ischemia-reperfusion injury is another indication. Endothelial celldysfunction may be important in both the initiation of, and inregulation of the sequelae of events that occur followingischemia-reperfusion injury.

Rheumatoid arthritis is a further indication. Blood vessel growth andtargeting of inflammatory cells through the vasculature is an importantcomponent in the pathogenesis of rheumatoid and sero-negative forms ofarthritis.

VEGF-E polypeptide or its antagonist may also be administeredprophylactically to patients with cardiac hypertrophy, to prevent theprogression of the condition, and avoid sudden death, including death ofasymptomatic patients. Such preventative therapy is particularlywarranted in the case of patients diagnosed with massive leftventricular cardiac hypertrophy (a maximal wall thickness of 35 mm ormore in adults, or a comparable value in children), or in instances whenthe hemodynamic burden on the heart is particularly strong.

VEGF-E polypeptide or its antagonist may also be useful in themanagement of atrial fibrillation, which develops in a substantialportion of patients diagnosed with hypertrophic cardiomyopathy.

Further indications include angina, myocardial infarctions such as acutemyocardial infarctions, and heart failure such as congestive heartfailure. Additional non-neoplastic conditions include psoriasis,diabetic and other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, thyroidhyperplasias (including Grave's disease), corneal and other tissuetransplantation, chronic inflammation, lung inflammation, nephroticsyndrome, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

In view of the above, the VEGF-E polypeptides or agonists or antagoniststhereof described herein, which are shown to alter or impact endothelialcell function, proliferation, and/or form, are likely to play animportant role in the etiology and pathogenesis of many or all of thedisorders noted above, and as such can serve as therapeutic targets toaugment or inhibit these processes or for vascular-related drugtargeting in these disorders.

12. Administration Protocols, Schedules, Doses, and Formulations

The molecules herein and agonists and antagonists thereto arepharmaceutically useful as a prophylactic and therapeutic agent forvarious disorders and diseases as set forth above.

The VEGF-E of the present invention can be formulated according to knownmethods to prepare pharmaceutically-useful compositions, whereby theVEGF-E hereof is combined in admixture with a pharmaceuticallyacceptable carrier vehicle. Suitable carrier vehicles and theirformulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in Remington's PharmaceuticalSciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. TheVEGF-E herein may be administered parenterally to subjects sufferingfrom cardiovascular diseases or conditions, or by other methods thatensure its delivery to the bloodstream in an effective form.

Compositions particularly well suited for the clinical administration ofVEGF-E hereof employed in the practice of the present invention include,for example, sterile aqueous solutions, or sterile hydratable powderssuch as lyophilized protein. It is generally desirable to includefurther in the formulation an appropriate amount of a pharmaceuticallyacceptable salt, generally in an amount sufficient to render theformulation isotonic. A pH regulator such as arginine base, andphosphoric acid, are also typically included in sufficient quantities tomaintain an appropriate pH, generally from 5.5 to 7.5. Moreover, forimprovement of shelf-life or stability of aqueous formulations, it mayalso be desirable to include further agents such as glycerol. In thismanner, variant VEGF-E formulations are rendered appropriate forparenteral administration, and, in particular, intravenousadministration.

Therapeutic compositions of the VEGF-E polypeptides or agonists orantagonists are prepared for storage by mixing the desired moleculehaving the appropriate degree of purity with optional pharmaceuticallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, 16th edition, Oslo, A. ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Additional examples of such carriers include ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts, or electrolytes such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and polyethylene glycol.Carriers for topical or gel-based forms of antagonist includepolysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. For all administrations, conventional depot formsare suitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations. The VEGF-Epolypeptides or agonists or antagonists will typically be formulated insuch vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.

Another formulation comprises incorporating a VEGF-E polypeptide orantagonist thereof into formed articles. Such articles can be used inmodulating endothelial cell growth and angiogenesis. In addition, tumorinvasion and metastasis may be modulated with these articles.

The VEGF-E to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). The VEGF-E ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of theVEGF-E preparations typically will be about from 6 to 8, although higheror lower pH values may also be appropriate in certain instances. It willbe understood that use of certain of the foregoing excipients, carriers,or stabilizers will result in the formation of salts of the VEGF-E.

An isotonifier may be present to ensure isotonicity of a liquidcomposition of the VEGF-E polypeptide or antagonist thereto, andincludes polyhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, andmannitol. These sugar alcohols can be used alone or in combination.Alternatively, sodium chloride or other appropriate inorganic salts maybe used to render the solutions isotonic.

The buffer may, for example, be an acetate, citrate, succinate, orphosphate buffer depending on the pH desired. The pH of one type ofliquid formulation of this invention is buffered in the range of about 4to 8, preferably about physiological pH.

The preservatives phenol, benzyl alcohol and benzethonium halides, e.g.,chloride, are known antimicrobial agents that may be employed.

Therapeutic VEGF-E polypeptide compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle. The formulations are preferably administered asrepeated intravenous (i.v.), subcutaneous (s.c.), or intramuscular(i.m.) injections, or as aerosol formulations suitable for intranasal orintrapulmonary delivery (for intrapulmonary delivery see, e.g., EP257,956).

VEGF-E polypeptide can also be administered in the form ofsustained-released preparations. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the protein, which matrices are in the form ofshaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (e.g.,poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.Biomed. Mater. Res. 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the Lupron Depot□(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid (EP133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions. Sustained-release VEGF-E polypeptidecompositions also include liposomally entrapped VEGF-E polypeptide.Liposomes containing VEGF-E polypeptide are prepared by methods knownper se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal therapy.

The therapeutically effective dose of VEGF-E polypeptide or antagonistthereto will, of course, vary depending on such factors as thepathological condition to be treated (including prevention), the methodof administration, the type of compound being used for treatment, anyco-therapy involved, the patient's age, weight, general medicalcondition, medical history, etc., and its determination is well withinthe skill of a practicing physician. Accordingly, it will be necessaryfor the therapist to titer the dosage and modify the route ofadministration as required to obtain the maximal therapeutic effect. Ifthe VEGF-E polypeptide has a narrow host range, for the treatment ofhuman patients formulations comprising human VEGF-E polypeptide, morepreferably native-sequence human VEGF-E polypeptide, are preferred. Theclinician will administer VEGF-E polypeptide until a dosage is reachedthat achieves the desired effect for treatment of the condition inquestion. For example, if the objective is the treatment of CHF, theamount would be one that inhibits the progressive cardiac hypertrophyassociated with this condition. The progress of this therapy is easilymonitored by echo cardiography. Similarly, in patients with hypertrophiccardiomyopathy, VEGF-E polypeptide can be administered on an empiricalbasis.

With the above guidelines, the effective dose generally is within therange of from about 0.001 to about 1.0 mg/kg, more preferably about0.01-1 mg/kg, most preferably about 0.01-0.1 mg/kg.

For non-oral use in treating human adult hypertension, it isadvantageous to administer VEGF-E polypeptide in the form of aninjection at about 0.01 to 50 mg, preferably about 0.05 to 20 mg, mostpreferably 1 to 20 mg, per kg body weight, 1 to 3 times daily byintravenous injection. For oral administration, a molecule based on theVEGF-E polypeptide is preferably administered at about 5 mg to 1 g,preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily.It should be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less than 0.5 ng/mg protein.Moreover, for human administration, the formulations preferably meetsterility, pyrogenicity, general safety, and purity as required by FDAOffice and Biologics standards.

The dosage regimen of a pharmaceutical composition containing VEGF-Epolypeptide to be used in tissue regeneration will be determined by theattending physician considering various factors that modify the actionof the polypeptides, e.g., amount of tissue weight desired to be formed,the site of damage, the condition of the damaged tissue, the size of awound, type of damaged tissue (e.g., bone), the patient's age, sex, anddiet, the severity of any infection, time of administration, and otherclinical factors. The dosage may vary with the type of matrix used inthe reconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF-I, to the final composition may also affectthe dosage. Progress can be monitored by periodic assessment oftissue/bone growth and/or repair, for example, X-rays, histomorphometricdeterminations, and tetracycline labeling.

The route of VEGF-E polypeptide or antagonist or agonist administrationis in accord with known methods, e.g., by injection or infusion byintravenous, intramuscular, intracerebral, intraperitoneal,intracerobrospinal, subcutaneous, intraocular, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation routes, or bysustained-release systems as noted below. The VEGF-E polypeptide orantagonists thereof also are suitably administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. The intraperitoneal route isexpected to be particularly useful, for example, in the treatment ofovarian tumors.

If a peptide or small molecule is employed as an antagonist or agonist,it is preferably administered orally or non-orally in the form of aliquid or solid to mammals.

Examples of pharmacologically acceptable salts of molecules that formsalts and are useful hereunder include alkali metal salts (e.g., sodiumsalt, potassium salt), alkaline earth metal salts (e.g., calcium salt,magnesium salt), ammonium salts, organic base salts (e.g., pyridinesalt, triethylamine salt), inorganic acid salts (e.g., hydrochloride,sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate,p-toluenesulfonate).

For compositions herein that are useful for bone, cartilage, tendon, orligament regeneration, the therapeutic method includes administering thecomposition topically, systemically, or locally as an implant or device.When administered, the therapeutic composition for use is in apyrogen-free, physiologically acceptable form. Further, the compositionmay desirably be encapsulated or injected in a viscous form for deliveryto the site of bone, cartilage, or tissue damage. Topical administrationmay be suitable for wound healing and tissue repair. Preferably, forbone and/or cartilage formation, the composition would include a matrixcapable of delivering the protein-containing composition to the site ofbone and/or cartilage damage, providing a structure for the developingbone and cartilage and preferably capable of being resorbed into thebody. Such matrices may be formed of materials presently in use forother implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradabiity, mechanical properties, cosmetic appearance, andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid,polyglycolic acid, and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above-mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability.

One specific embodiment is a 50:50 (mole weight) copolymer of lacticacid and glycolic acid in the form of porous particles having diametersranging from 150 to 800 microns. In some applications, it will be usefulto utilize a sequestering agent, such as carboxymethyl cellulose orautologous blood clot, to prevent the polypeptide compositions fromdisassociating from the matrix.

One suitable family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydoxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, andcarboxymethylcellulose, one preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer, and poly(vinyl alcohol).The amount of sequestering agent useful herein is 0.5-20 wt %,preferably 1-10 wt %, based on total formulation weight, whichrepresents the amount necessary to prevent desorption of the polypeptide(or its antagonist) from the polymer matrix and to provide appropriatehandling of the composition, yet not so much that the progenitor cellsare prevented from infiltrating the matrix, thereby providing thepolypeptide (or its antagonist) the opportunity to assist the osteogenicactivity of the progenitor cells.

Generally, where the disorder permits, one should formulate and dose theVEGF-E for site-specific delivery. This is convenient in the case ofwounds and ulcers.

When applied topically, the VEGF-E is suitably combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of such other ingredients, except that they must bepharmaceutically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

For obtaining a gel formulation, the VEGF-E formulated in a liquidcomposition may be mixed with an effective amount of a water-solublepolysaccharide or synthetic polymer such as polyethylene glycol to forma gel of the proper viscosity to be applied topically. Thepolysaccharide that may be used includes, for example, cellulosederivatives such as etherified cellulose derivatives, including alkylcelluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses,for example, methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose;starch and fractionated starch; agar; alginic acid and alginates; gumarabic; pullullan; agarose; carrageenan; dextrans; dextrins; fructans;inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans; andsynthetic biopolymers; as well as gums such as xanthan gum; guar gum;locust bean gum; gum arabic; tragacanth gum; and karaya gum; andderivatives and mixtures thereof. The preferred gelling agent herein isone that is inert to biological systems, nontoxic, simple to prepare,and not too runny or viscous, and will not destabilize the VEGF-E heldwithin it.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture oflow- and high-molecular-weight polyethylene glycols to obtain the properviscosity. For example, a mixture of a polyethylene glycol of molecularweight 400-600 with one of molecular weight 1500 would be effective forthis purpose when mixed in the proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides andpolyethylene glycols is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient. Additionally, the cellulose derivatives may be in the formof alkali metal salts, for example, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2-5%, more preferably about 3%, of the gel and the VEGF-E is present inan amount of about 300-1000 mg per ml of gel.

13. Combination Therapies

The effectiveness of the VEGF-E polypeptide or an agonist or antagonistthereof in preventing or treating the disorder in question may beimproved by administering the active agent serially or in combinationwith another agent that is effective for those purposes, either in thesame composition or as separate compositions. Hence, it is within thescope hereof to combine the VEGF-E therapy with other novel orconventional therapies (e.g., growth factors such as VEGF, aFGF, bFGF,PDGF, IGF, NGF, anabolic steroids, EGF or TGF-alpha) for enhancing theactivity of any of the growth factors, including VEGF-E, in promotingcell proliferation, survival, differentiation, and repair. It is notnecessary that such cotreatment drugs be included per se in thecompositions of this invention, although this will be convenient wheresuch drugs are proteinaceous. Such admixtures are suitably administeredin the same manner and for the same purposes as the VEGF-E used alone.

For treatment of cardiac hypertrophy, VEGF-E polypeptide therapy can becombined with the administration of inhibitors of known cardiac myocytehypertrophy factors, e.g., inhibitors of α-adrenergic agonists such asphenylephrine; endothelin-1 inhibitors such as BOSENTAN™ and MOXONODIN™;inhibitors to CT-1 (U.S. Pat. No. 5,679,545); inhibitors to LIF; ACEinhibitors; des-aspartate-angiotensin I inhibitors (U.S. Pat. No.5,773,415), and angiotensin II inhibitors.

For treatment of cardiac hypertrophy associated with hypertension,VEGF-E polypeptide can be administered in combination with β-adrenergicreceptor blocking agents, e.g., propranolol, timolol, tertalolol,carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol,metoprolol, or carvedilol; ACE inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, or lisinopril; diuretics,e.g., chorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, orindapamide; and/or calcium channel blockers, e.g., diltiazem,nifedipine, verapamil, or nicardipine. Pharmaceutical compositionscomprising the therapeutic agents identified herein by their genericVEGF-Es are commercially available, and are to be administered followingthe manufacturers' instructions for dosage, administration, adverseeffects, contraindications, etc. See, e.g., Physicians' Desk Reference(Medical Economics Data Production Co.: Montvale, N.J., 1997), 51thEdition.

Preferred candidates for combination therapy in the treatment ofhypertrophic cardiomyopathy are β-adrenergic-blocking drugs (e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol),verapamil, difedipine, or diltiazem. Treatment of hypertrophy associatedwith high blood pressure may require the use of antihypertensive drugtherapy, using calcium channel blockers, e.g., diltiazem, nifedipine,verapamil, or nicardipine; β-adrenergic blocking agents; diuretics,e.g., chorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, orindapamide; and/or ACE-inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, or lisinopril.

For other indications, VEGF-E polypeptides or their antagonists may becombined with other agents beneficial to the treatment of the boneand/or cartilage defect, wound, or tissue in question. These agentsinclude various growth factors such as EGF, PDGF, TGF-α or TGF-β, IGF,FGF, and CTGF.

In addition, VEGF-E polypeptides or their antagonists used to treatcancer may be combined with cytotoxic, chemotherapeutic, orgrowth-inhibitory agents as identified above. Also, for cancertreatment, the VEGF-E polypeptide or antagonist thereof is suitablyadministered serially or in combination with radiological treatments,whether involving irradiation or administration of radioactivesubstances.

The effective amounts of the therapeutic agents administered incombination with VEGF-E polypeptide or antagonist thereof will be at thephysician's or veterinarian's discretion. Dosage administration andadjustment is done to achieve maximal management of the conditions to betreated. For example, for treating hypertension, these amounts ideallytake into account use of diuretics or digitalis, and conditions such ashyper- or hypotension, renal impairment, etc. The dose will additionallydepend on such factors as the type of the therapeutic agent to be usedand the specific patient being treated. Typically, the amount employedwill be the same dose as that used, if the given therapeutic agent isadministered without VEGF-E polypeptide. A useful molar ratio of VEGF-Eto secondary growth factors is typically 1:0.1-10, with about equimolaramounts being preferred.

14. Articles of Manufacture

An article of manufacture such as a kit containing VEGF-E polypeptide orantagonists thereof useful for the diagnosis or treatment of thedisorders described above comprises at least a container and a label.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition that iseffective for diagnosing or treating the condition and may have asterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is the VEGF-Epolypeptide or an agonist or antagonist thereto. The label on, orassociated with, the container indicates that the composition is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. The article of manufacture may also comprisea second or third container with another active agent as describedabove.

F. Anti-VEGF-E Antibodies

The present invention further provides anti-VEGF-E polypeptideantibodies. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-VEGF-E antibodies of the present invention may comprisepolyclonal antibodies. Methods of preparing polyclonal antibodies areknown to the skilled artisan. Polyclonal antibodies can be raised in amammal, for example, by one or more injections of an immunizing agentand, if desired, an adjuvant. Typically, the immunizing agent and/oradjuvant will be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include the VEGF-Epolypeptide or a fusion protein thereof. It may be useful to conjugatethe immunizing agent to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

2. Monoclonal Antibodies

The anti-VEGF-E antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the VEGF-E polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine, and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against aVEGF-E polypeptide. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy-chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart.

3. Humanized Antibodies

The anti-VEGF-E antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂, or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary-determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat, or rabbit having the desired specificity, affinity,and capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aVEGF-E polypeptide, the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide-exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992)and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989).

G. Uses for Anti-VEGF-E Antibodies

The anti-VEGF-E antibodies of the present invention have variousutilities. For example, anti-VEGF-E antibodies may be used in diagnosticassays for VEGF-E polypeptides, e.g., detecting expression in specificcells, tissues, or serum. Various diagnostic assay techniques known inthe art may be used, such as competitive binding assays, direct orindirect sandwich assays and immunoprecipitation assays conducted ineither heterogeneous or homogeneous phases (Zola, Monoclonal Antibodies:A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158). Theantibodies used in the diagnostic assays can be labeled with adetectable moiety. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase. Any methodknown in the art for conjugating the antibody to the detectable moietymay be employed, including those methods described by Hunter et al.,Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Painet al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-VEGF-E antibodies also are useful for the affinity purification ofVEGF-E polypeptides from recombinant cell culture or natural sources. Inthis process, the antibodies against a VEGF-E polypeptide areimmobilized on a suitable support, such as Sephadex™ resin or filterpaper, using methods well known in the art. The immobilized antibodythen is contacted with a sample containing the VEGF-E polypeptide to bepurified, and thereafter the support is washed with a suitable solventthat will remove substantially all the material in the sample except theVEGF-E polypeptide, which is bound to the immobilized antibody. Finally,the support is washed with another suitable solvent that will releasethe VEGF-E polypeptide from the antibody.

1. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a VEGF-E polypeptide identified herein,as well as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersas noted above and below in the form of pharmaceutical compositions.

If the VEGF-E polypeptide is intracellular and whole antibodies are usedas inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

2. Methods of Treatment using the Antibody

It is contemplated that the antibodies to VEGF-E polypeptide may be usedto treat various cardiovascular, endothelial, and angiogenic conditionsas noted above.

The antibodies are administered to a mammal, preferably a human, inaccord with known methods, such as intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous administration of the antibody is preferred.

Other therapeutic regimens may be combined with the administration ofthe antibodies of the instant invention as noted above. For example, ifthe antibodies are to treat cancer, the patient to be treated with suchantibodies may also receive radiation therapy. Alternatively, or inaddition, a chemotherapeutic agent may be administered to the patient.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers' instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService, Ed., M. C. Perry (Williams & Wilkins: Baltimore, Md., 1992).The chemotherapeutic agent may precede, or follow administration of theantibody, or may be given simultaneously therewith. The antibody may becombined with an anti-oestrogen compound such as tamoxifen or EVISTA™ oran anti-progesterone such as onapristone (see, EP 616812) in dosagesknown for such molecules.

If the antibodies are used for treating cancer, it may be desirable alsoto administer antibodies against other tumor-associated antigens, suchas antibodies that bind to one or more of the ErbB2, EGFR, ErbB3, ErbB4,or VEGF receptor(s). These also include the agents set forth above.Also, the antibody is suitably administered serially or in combinationwith radiological treatments, whether involving irradiation oradministration of radioactive substances. Alternatively, or in addition,two or more antibodies binding the same or two or more differentantigens disclosed herein may be co-administered to the patient.Sometimes, it may be beneficial also to administer one or more cytokinesto the patient. In a preferred embodiment, the antibodies herein areco-administered with a growth-inhibitory agent. For example, thegrowth-inhibitory agent may be administered first, followed by anantibody of the present invention. However, simultaneous administrationor administration of the antibody of the present invention first is alsocontemplated. Suitable dosages for the growth-inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth-inhibitory agent and the antibody herein.

In one embodiment, vascularization of tumors is attacked in combinationtherapy. The anti-VEGF-E polypeptide and another antibody (e.g.,anti-VEGF) are administered to tumor-bearing patients at therapeuticallyeffective doses as determined, for example, by observing necrosis of thetumor or its metastatic foci, if any. This therapy is continued untilsuch time as no further beneficial effect is observed or clinicalexamination shows no trace of the tumor or any metastatic foci. Then TNFis administered, alone or in combination with an auxiliary agent such asalpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin,anti-heregulin antibody, D-factor, interleukin-1 (IL-1), interleukin-2(IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), oragents that promote microvascular coagulation in tumors, such asanti-protein C antibody, anti-protein S antibody, or C4b binding protein(see WO 91/01753, published 21 Feb. 1991), or heat or radiation.

Since the auxiliary agents will vary in their effectiveness, it isdesirable to compare their impact on the tumor by matrix screening inconventional fashion. The administration of anti-VEGF-E polypeptideantibody and TNF is repeated until the desired clinical effect isachieved. Alternatively, the anti-VEGF-E polypeptide antibody isadministered together with TNF and, optionally, auxiliary agent(s). Ininstances where solid tumors are found in the limbs or in otherlocations susceptible to isolation from the general circulation, thetherapeutic agents described herein are administered to the isolatedtumor or organ. In other embodiments, a FGF or PDGF antagonist, such asan anti-FGF or an anti-PDGF neutralizing antibody, is administered tothe patient in conjunction with the anti-VEGF-E polypeptide antibody.Treatment with anti-VEGF-E polypeptide antibodies preferably may besuspended during periods of wound healing or desirableneovascularization.

For the prevention or treatment of cardiovascular, endothelial, andangiogenic disorder, the appropriate dosage of an antibody herein willdepend on the type of disorder to be treated, as defined above, theseverity and course of the disease, whether the antibody is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments.

For example, depending on the type and severity of the disorder, about 1μg/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily or weekly dosage might range from about 1μg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is repeated or sustained until a desiredsuppression of disorder symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays, including, for example, radiographictumor imaging.

3. Articles of Manufacture with Antibodies

An article of manufacture containing a container with the antibody and alabel is also provided. Such articles are described above, wherein theactive agent is an anti-VEGF-E antibody.

4. Diagnosis and Prognosis of Tumors using Antibodies

If the indication for which the antibodies are used is cancer, whilecell-surface proteins, such as growth receptors overexpressed in certaintumors, are excellent targets for drug candidates or tumor (e.g.,cancer) treatment, the same proteins along with VEGF-E polypeptides findadditional use in the diagnosis and prognosis of tumors. For example,antibodies directed against the VEGF-E polypeptides may be used as tumordiagnostics or prognostics.

For example, antibodies, including antibody fragments, can be usedqualitatively or quantitatively to detect the expression of genesincluding the gene encoding the VEGF-E polypeptide. The antibodypreferably is equipped with a detectable, e.g., fluorescent label, andbinding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. Such binding assaysare performed essentially as described above.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent to those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example I Identification of Clones Encoding a VEGF-Related Protein(VEGF-E)

Probes based on an expressed sequence tag (EST) identified from theIncyte Pharmaceuticals database due to homology with VEGF were used toscreen a cDNA library derived from the human glioma cell line G61. Inparticular, Incyte Clone “INC1302516” was used to generate the followingfour probes: (SEQ ID NO:3) 5′-ACTTCTCAGTGTCCATAAGGG; (SEQ ID NO:4)5′-GAACTAAAGAGAACCGATACCATTTTCTGGCCAGGTTGTC; (SEQ ID NO:5)5′-CACCACAGCGTTTAACCAGG; and (SEQ ID NO:6) 5′-ACAACAGGCACAGTTCCCAC.

Nine positives were identified and characterized. Three clones containedthe full coding region and were identical in sequence. Partial cloneswere also identified from a fetal lung library and were identical withthe glioma-derived sequence with the exception of one nucleotide change,which did not alter the encoded amino acid.

Example 2 Expression Constructs

For mammalian protein expression, the entire open reading frame (ORF)was cloned into a CMV-based expression vector. An epitope-tag (FLAG™,Kodak) and Histidine-tag (His8) were inserted between the ORF and stopcodon. VEGF-E-His8 and VEGF-E-FLAG were transfected into human embryonickidney 293 cells by SuperFec™ (Qiagen) and pulse-labeled for 3 hourswith (³⁵S)methionine and (³⁵C)cysteine. Both epitope-tagged proteinsco-migrate when 20 microliters of 15-fold concentrated serum-freeconditioned medium were electrophoresed on a polyacrylamide gel (Novex)in sodium dodecyl sulfate sample buffer (SDS-PAGE). The VEGF-E-IgGexpression plasmid was constructed by cloning the ORF in front of thehuman Fc (IgG) sequence.

The VEGF-E-IgG plasmid was co-transfected with Baculogold Baculovirus™DNA (Pharmingen) using Lipofectin™ (GibcoBRL) into 10⁵ Sf9 cells grownin Hink's™ TNM-FH medium (JRH Biosciences) supplemented with 10% fetalbovine serum. Cells were incubated for 5 days at 28° C. The supernatantwas harvested and subsequently used for the first viral amplification byinfecting Sf9 cells at an approximate multiplicity of infection (MOI) of10. Cells were incubated for 3 days, then supernatant was harvested, andexpression of the recombinant plasmid was determined by binding of 1 mlof supernatant to 30 μl of Protein-A Sepharose™ CL-4B beads (Pharmacia)followed by subsequent SDS-PAGE analysis. The first amplificationsupernatant was used to infect a 500 ml spinner culture of Sf9 cellsgrown in ESF-921 medium (Expression Systems LLC) at an approximate MOIof 0.1. Cells were treated as above, except harvested supernatant wassterile filtered. Specific protein was purified by binding to Protein-ASepharose 4 Fast Flow™ (Pharmacia) column.

Example 3 Northern Blot Analyses

Blots of human poly(A)+ RNA from multiple adult and fetal tissues andtumor cell lines were obtained from Clontech (Palo Alto, Calif.).Hybridization was carried out using ³²P-labeled probes containing theentire coding region and washed in 0.1×SSC, 0.1% SDS at 63° C.

VEGF-E mRNA was detectable in fetal lung, kidney, brain, and liver andin adult heart, placenta, liver, skeletal muscle, kidney, and pancreas.VEGF-E mRNA was also found in A549 lung adenocarcinoma and HeLa cervicaladenocarcinoma cell lines.

Example 4 In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis, and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1: 169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A (³³-P)UTP-labeled antisense riboprobe was generatedfrom a PCR product of 980 bp (using the oligonucleotide primersindicated below) and hybridized at 55° C. overnight. The slides weredipped in KODAK NTB2™ nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed-vacuum dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl RNAsin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. A total of 1.0 μl RQ1DNase was added, followed by incubation at 37° C. for 15 minutes. Atotal of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, andthe mixture was pipetted onto DE81 paper. The remaining solution wasloaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10(6 minutes). The filtration unit was inverted over a second tube andspun using program 2 (3 minutes). After the final recovery spin, a totalof 100 μl TE was added. Then 1 μl of the final product was pipetted onDE81 paper and counted in 6 ml of BIOFLUOR II™.

The probe was run on a TBE/urea gel. A total of 1-3 μl of the probe or 5μl of RNA Mrk III was added to 3 μl of loading buffer. After heating ona 95° C. heat block for three minutes, the gel was immediately placed onice. The wells of gel were flushed, and the sample was loaded and run at180-250 volts for 45 minutes. The gel was wrapped in plastic wrap(SARAN™ brand) and exposed to XAR film with an intensifying screen in a−70° C. freezer one hour to overnight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminum trays, andthawed at room temperature for 5 minutes. The trays were placed in a 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml s.c. H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNAse-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,and 100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-Embedded Sections

The slides were deparaffinized, placed in s.c. H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNAse-free RNAse buffer; 37° C., 15 minutes) for human embryo tissue, or8× proteinase K (100 μl in 250 ml RNAse buffer, 37° C., 30 minutes) forformalin tissues. Subsequent rinsing in 0.5×SSC and dehydration wereperformed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide). The filter paper was saturated. The tissue was coveredwith 50 μl of hybridization buffer (3.75 g dextran sulfate+6 ml s.c.H₂O), vortexed, and heated in the microwave for 2 minutes with the caploosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC, and9 ml s.c. H₂O were added, and the tissue was vortexed well and incubatedat 42° C. for 1-4 hours.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer was added per slide. After vortexing, 50 μl ³³P mixwas added to 50 μl prehybridization on the slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done for 2×10 minutes with 2×SSC, EDTA at room temperature(400 ml 20×SSC+16 ml 0.25 M EDTA, V_(f)=4L), followed by RNAseAtreatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml RNAsebuffer=20 μg/ml). The slides were washed 2×10 minutes with 2×SSC, EDTAat room temperature. The stringency wash conditions were as follows: 2hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4L).

F. Oligonucleotide Primers

In situ analysis was performed on the DNA29101 sequence disclosedherein. The oligonucleotide primers employed to prepare the riboprobefor these analyses were as follows. p1: (SEQ ID NO:7) 5′-GGA TTC TAA TACGAC TCA CTA TAG GGC GGC GGA ATC CAA CCT GAG TAG p2 (SEQ ID NO:8) 5′-CTATGA AAT TAA CCC TCA CTA AAG GGA GCG GCT ATC CTC CTG TGC TC

G. Results

The results from this in situ analysis were as follows.

For the lower human fetal limb, there was expression of VEGF-E indeveloping lower limb bones at the edge of the cartilagenous anlage(i.e., around the outside edge), in developing tendons, in vascularsmooth muscle, and in cells embracing developing skeletal musclemyocytes and myotubes. Expression was also observed at the epiphysealgrowth plate. There was human fetal lymph node expression of VEGF-E inthe marginal sinus of developing lymph nodes. There was human fetalthymus expression in the subcapsular region of the thymic cortex,possibly representing either the subcapsular epithelial cells or theproliferating, double-negative thymocytes that are found in this region.The human fetal spleen was negative for expression.

Trachea expression of VEGF-E in the smooth muscle of human fetal tissuewas observed. There was human fetal brain (cerebral cortex) focalexpression of VEGF-E in cortical neurons. The human fetal spinal cordwas negative. There was human fetal small intestine expression of VEGF-Ein smooth muscle. In addition, there was human fetal thyroid generalizedexpression of VEGF-E over thryoid epithelium. The human fetal adrenalgland was negative. Liver expression of VEGF-E in human fetal ductalplate cells was observed, as well as human fetal stomach expression inmural smooth muscle and human fetal skin expression in basal layer ofthe squamous epithelium. In addition, there was human fetal placentaexpression of VEGF-E in interstitial cells in trophoblastic villi, andhuman fetal cord expression in the wall of the arteries and veins.

When tested in superovulated rat ovaries, all sections, control andsuperovulated ovaries, were negative with both antisense and senseprobes. Either the message was not expressed in this model, or the humanprobe does not cross react with rat.

High expression of VEGF-E was observed at the following additionalsites:

chimp ovary—granulosa cells of maturing follicles, lower intensitysignal observed over thecal cells.

chimp parathyroid—high expression over chief cells.

human fetal testis—moderate expression over stromal cells surroundingdeveloping tubules

human fetal lung—high expression over chondrocytes in developingbronchial tree, and low level expression over branching bronchialepithelium.

Specific expression was not observed over the renal cell, gastric andcolonic carcinomas.

The fetal tissues examined in the above study (E12-E16 weeks) included:placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,heart, great vessels, oesophagus, stomach, small intestine, spleen,thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lowerlimb.

The adult tissues examined in the above study included:

liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas,lung, skin, cerebral cortex (rm), hippocampus (rm), cerebellum (rm),penis, eye, bladder, stomach, gastric carcinoma, colon, coloniccarcinoma, and chondrosarcoma, as well as tissues havingacetominophen-induced liver injury, and hepatic cirrhosis.

In summary, the expression pattern suggests that VEGF-E may be involvedin cell differentiation and/or proliferation. Expression patterns indeveloping skeletal muscle suggest that the protein may be involved inmyoblast differentiation and/or proliferation.

Example 5 Myocyte Hypertrophy Assay

Myocytes from neonatal Harlan Sprague Dawley rat heart ventricle (23days gestation) were plated in duplicate at 75000 cells/ml in a 96-wellplate. Cells were treated for 48 h with 2000, 200, 20, or 2 ng/mlVEGF-E-IgG. Myocytes were stained with crystal violet to visualizemorphology and scored on a scale of 3 to 7, 3 being nonstimulated and 7being full-blown hypertrophy. 2000 ng/ml and 200 ng/ml VEGF-E causedhypertrophy, scored as a 5.

Example 6 Cell Proliferation Assay

Mouse embryonic fibroblast C₃HlOT1/2 cells (ATCC) were grown in 50:50Ham's F-12: low glucose DMEM medium containing 10% fetal calf serum(FCS). Cells were plated in duplicate in a 24-well plate at 1000, 2000,and 4000 cells/well. After 48 hours, cells were switched to mediumcontaining 2% FCS and were incubated for 72 hours with 200, 800, or 2000ng/ml VEGF-E or no growth factor added.

Approximately 1.5 fold greater number of cells were measured in thepresence of 200 ng/ml VEGF-E as in its absence, at all three celldensities.

Example 7 Endothelial Cell Survival Assay

Human umbilical vein endothelial cells (HUVEC, Cell Systems) weremaintained in Complete Media (Cell Systems) and plated in triplicate inserum-free medium (Basic Media from Cell Systems containing 0.1% BSA) at20,000 cells/well of a 48-well plate. Cells were incubated for 5 dayswith 200 or 400 ng/ml VEGF-E-IgG, 100 ng/ml VEGF, 20 ng/ml basic FGF, orno addition.

Survival was 2-3 times greater with VEGF-E as compared to lack of growthfactor addition. VEGF and basic FGF were included as positive controls.

Example 8 Stimulation of Endothelial Tube Formation

This assay follows the assay described in Davis and Camarillo,Experimental Cell Research, 224:39-51 (1996), or one modified from it asfollows:

Protocol: HUVEC cells (passage number less than 8 from primary) aremixed with type I rat tail collagen, final concentration 2.6 mg/ml at adensity of 6×10⁵ cells/mil and plated at 50 μl per well on a 96-wellplate. The gel is allowed to solidify for 1 hr at 37° C., then 50 μl perwell of M199 culture media supplemented with 1% FBS and a VEGF-E sample(at dilutions of 1%, 0.1%, and 0.01%, respectively) is added along with1 μM 6-FAM-FITC dye to stain vacuoles while they are forming. Cells areincubated at 37° C./5% CO₂ for 48 hr, fixed with 3.7% formalin at roomtemperature for 10 minutes, washed with PBS five times, then stainedwith Rh-Phalloidin at 4° C. overnight followed by nuclear staining with4 μM DAPI.

1. Apoptosis Assay

This assay will identify factors that facilitate cell survival in a3-dimensional matrix in the presence of exogenous growth factors (VEGF,bFGF without PMA).

A positive result is equal to or less than 1. 0=no apoptosis, 1=lessthan 20% cells are apoptotic, 2=less than 50% cells are apoptotic,3=greater than 50% cells are apoptotic. Stimulators of apoptosis in thissystem are expected to be apoptotic factors, and inhibitors are expectedto prevent or lessen apoptosis.

2. Vacuoles Assay

This assay will identify factors that stimulate endothelial vacuoleformation and lumen formation in the presence of bFGF and VEGF (40ng/ml).

A positive result is equal to or greater than 2. 1=vacuoles present inless than 20% of cells, 2=vacuoles present in 20-50% of cells,3=vacuoles present in greater than 50% of cells. This assay is designedto identify factors that are involved in stimulating pinocytosis, ionpumping, permeability, and junction formation.

3. Tube Formation Assay

This assay is to identify factors that stimulate endothelial tubeformation in a 3-dimensional matrix. This assay will identify factorsthat stimulate endothelial cells to differentiate into a tube-likestructure in a 3-dimensional matrix in the presence of exogenous growthfactors (VEGF, bFGF).

A positive result is equal to or greater than 2. 1=cells are all round,2=cells are elongated, 3=cells are forming tubes with some connections,4=cells are forming complex tubular networks. This assay would identifyfactors that may be involved in stimulating tracking, chemotaxis, orendothelial shape change.

The results are shown in FIGS. 3 through 5. FIG. 3A shows the HUVEC tubeformation when no growth factors are present. FIG. 3B shows whereVEGF/bFGF, and PMA are present, FIG. 3C shows where VEGF and bFGF arepresent, FIG. 3D shows where VEGF and PMA are present, FIG. 3E showswhere bFGF and PMA are present, FIG. 3F shows where VEGF is present,FIG. 3G shows where bFGF is present, and FIG. 3H shows where PMA ispresent.

FIGS. 4A and 4B show, respectively, the effect on HUVEC tube formationof VEGF-E-IgG at 1% dilution and of a buffer control (10 mM HEPES/0.14MNaCl/4% mannitol, pH 6.8) at 1% dilution. FIGS. 5A and 5B show,respectively, the effect on HUVEC tube formation of VEGF-E-poly-his at1% dilution and of the buffer control used for VEGF-E-IgG at 1%dilution.

The results clearly show more complex tube formation with the VEGF-E-IgGand VEGF-E-poly-his samples than with the buffer controls.

Example 9 Transgenic Mice

Transgenic mice were generated by microinjection of C57B1/6/SJL F2 mouseembryos (DNAX) with a vector suitable for such microinjection containingthe cDNA encoding VEGF-E under the control of a keratin promoter (Xie etal., Nature, 391: 90-92 (1998)), driving expression in the skin.

Transgenic pups were wrinkled and shiny at birth and were delayed ingetting their hair. The mice lost their phenotype by two weeks of age.There were no detectable histopathic changes.

Example 10 Production of Antibodies

Polyclonal antisera were generated in female New Zealand White rabbitsagainst human VEGF-E. The protein was homogenized with Freund's completeadjuvant for the primary injection and with Freund's incomplete adjuvantfor all subsequent boosts. For the primary immunization and the firstboost, 3.3 μg per kg body weight was injected directly into thepopliteal lymph nodes, according to Bennett et al., J. Biol. Chem., 266:23060-23067 (1991); and “Production of Antibodies by Inoculation intoLymph Nodes” by Sigel, Sinha and VanderLaan in Methods in Enzymology,Vol. 93 (New York: Academic Press, 1983). For all subsequent boosts, 3.3μg per kg body weight was injected into subcutaneous and intramuscularsites. Injections were done every 3 weeks with bleeds taken on thefollowing 2 weeks after each injection. The polyclonal antisera thusobtained contained antibodies binding VEGF-E, as revealed byimmunoprecipitation experiments.

Example 11 Inhibition of VEGF-Stimulated Endothelial Cell (ACE Cells)Growth

Bovine adrenal cortical capillary endothelial cells (ACE cells) (fromprimary culture, maximum of 12-14 passages) were plated in 96-wellplates at 500 cells/well per 100 microliter. Assay media included lowglucose DMEM, 10% calf serum, 2 mM glutamine, and 1×penicillin/streptomycin/fungizone. Control wells included the following:(1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/mlFGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGFplus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus 5 ng/mlLIF. The test sample, poly-his tagged VEGF-E polypeptide (described inthe Examples above; in 100 microliter volumes), was then added to thewells (at dilutions of 1%, 0.1% and 0.01%, respectively). The cellcultures were incubated for 6-7 days at 37□C/5% CO₂. After theincubation, the media in the wells was aspirated, and the cells werewashed 1× with PBS. An acid phosphatase reaction mixture (100microliter; 0.1M sodium acetate, pH 5.5, 0.1% Triton X-100, 10 mMp-nitrophenyl phosphate) was then added to each well. After a 2 hourincubation at 37° C., the reaction was stopped by addition of 10microliters 1N NaOH. Optical density (OD) was measured on a microplatereader at 405 nm.

The activity of VEGF-E was calculated as the percent inhibition of VEGF(3 ng/ml) stimulated proliferation (as determined by the acidphosphatase activity at OD 405 nm) relative to the cells withoutstimulation. TGF-beta was employed as an activity reference at 1 ng/ml,TGF-beta blocks 70-90% of VEGF-stimulated ACE cell proliferation.Results of the assay were interpreted as “positive” if the observedinhibition was ≧30%.

In a first assay run, the VEGF-E at 1%, 0.1%, and 0.01% dilutionsexhibited 52%, 90% and 96% inhibition, respectively. In a second assayrun, the VEGF-E at 1%, 0.1%, and 0.01% dilutions exhibited 57%, 93% and91% inhibition, respectively.

Deposit of Material

The following material has been deposited with the American Type CultureCollection, 10801 University Blvd., Manassas, Va. USA (ATCC): MaterialATCC Dep. No. Deposit Date DNA29101-1272 209653 Mar. 5, 1998

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe material on deposit should die or be lost or destroyed whencultivated under suitable conditions, the material will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated nucleic acid comprising a nucleotide sequence encoding avascular endothelial cell growth factor-E (VEGF-E) polypeptidecomprising amino acid residues 1 through 345 of FIG. 2 (SEQ ID NO:2). 2.The nucleic acid of claim 1 comprising the sequence of nucleotides 259through 1293 of FIG. 1 (SEQ ID NO:1), or its complement.
 3. An isolatednucleic acid comprising a nucleotide sequence which hybridizes understringent conditions to the nucleotide sequence of claim
 1. 4. A vectorcomprising the nucleic acid of claim
 1. 5. A host cell comprising thenucleic acid of claim
 1. 6. The host cell of claim 5, wherein said cellis a Chinese Hamster Ovary cell, an insect cell, an E. coli cell, or ayeast cell.
 7. The host cell of claim 6 that is a baculovirus-infectedinsect cell.
 8. A process for producing a vascular endothelial cellgrowth factor-E (VEGF-E) polypeptide comprising culturing the host cellof claim 5 under conditions suitable for expression of the VEGF-Epolypeptide and recovering the VEGF-E polypeptide from the cell culture.9. A polypeptide produced by the process of claim
 8. 10. A VEGF-Epolypeptide comprising amino acid residues 1 through 345 of FIG. 2 (SEQID NO:2).
 11. A VEGF-E polypeptide selected from the group consistingof: (a) a polypeptide comprising amino acid residues 1 through 345 ofFIG. 2 (SEQ ID NO:2); and (b) a polypeptide fragment of (a), whereinsaid fragment is biologically active.
 12. A VEGF-E polypeptide encodedby the nucleotide sequence insert of ATCC deposit No.
 209653. 13. Achimeric polypeptide comprising the polypeptide of claim 10 fused to aheterologous amino acid sequence.
 14. The chimeric polypeptide of claim13, wherein said heterologous amino acid sequence is an epitope tagsequence or a Fc region of an immunoglobulin.
 15. A compositioncomprising the polypeptide of claim 10 in admixture with a carrier. 16.The composition of claim 15 comprising a therapeutically effectiveamount of the polypeptide, wherein the carrier is a pharmaceuticallyacceptable carrier.
 17. A pharmaceutical product comprising: (a) thecomposition of claim 15; (b) a container containing said composition;and (c) a label affixed to said container, or a package insert includedin said pharmaceutical product, referring to the use of said VEGF-Epolypeptide in the treatment of a cardiovascular or endothelialdisorder.
 18. A method for treating a cardiovascular or endothelialdisorder in a mammal comprising administering to the mammal an effectiveamount of the composition of claim
 15. 19. The method of claim 18wherein the disorder is cardiac hypertrophy, trauma, or a bone-relateddisorder.
 20. The method of claim 19 wherein said cardiac hypertrophy ischaracterized by the presence of an elevated level of PGF_(2α).
 21. Themethod of claim 19 wherein said cardiac hypertrophy has been induced bymyocardial infarction.
 22. The method of claim 21 wherein said VEGF-Epolypeptide administration is initiated within 48 hours followingmyocardial infarction.
 23. A method for diagnosing a disease or asusceptibility to a disease related to a mutation in a nucleic acidsequence encoding vascular endothelial cell growth factor-E (VEGF-E)comprising: (a) isolating a nucleic acid sequence encoding VEGF-E from asample derived from a host; and (b) determining a mutation in thenucleic acid sequence encoding VEGF-E.
 24. A method of diagnosingcardiovascular and endothelial disorders in a mammal comprisingdetecting the level of expression of a gene encoding a vascularendothelial cell growth factor-E (VEGF-E) polypeptide (a) in a testsample of tissue cells obtained from the mammal, and (b) in a controlsample of known normal tissue cells of the same cell type, wherein ahigher or lower expression level in the test sample indicates thepresence of a cardiovascular or endothelial dysfunction in the mammalfrom which the test tissue cells were obtained.
 25. A method foridentifying an agonist to a vascular endothelial cell growth factor-E(VEGF-E) polypeptide comprising: (a) contacting cells and a candidatecompound under conditions that allow the polypeptide to stimulateproliferation of the cells; and (b) measuring the extent to which cellproliferation is inhibited by the compound.
 26. An agonist to a VEGF-Epolypeptide identified by the method of claim
 25. 27. A method foridentifying a compound that inhibits the expression or activity of avascular endothelial cell growth factor-E (VEGF-E) polypeptide,comprising: (a) contacting a candidate compound with the polypeptideunder conditions and for a time sufficient to allow the compound andpolypeptide to interact; and (b) measuring the extent to which thecompound interacts with the polypeptide.
 28. A compound identified bythe method of claim
 27. 29. An isolated antibody that binds a vascularendothelial cell growth factor-E (VEGF-E) polypeptide.
 30. The antibodyof claim 29 that is a monoclonal antibody.
 31. A method for determiningthe presence of a vascular endothelial cell growth factor-E (VEGF-E)polypeptide comprising exposing a cell suspected of containing theVEGF-E polypeptide to the antibody of claim 29 and determining bindingof said antibody to said cell.
 32. A method of diagnosingcardiovascular, endothelial, or angiogenic disorders in a mammalcomprising (a) contacting the antibody of claim 29 with a test sample oftissue cells obtained from the mammal, and (b) detecting the formationof a complex between the anti-VEGF-E antibody and the VEGF-E polypeptidein the test sample.
 33. An article of manufacture, comprising: acontainer; a label on the container; and a composition comprising theantibody of claim 29 contained within the container; wherein the labelon the container indicates instructions for using the antibody in atherapeutic method or diagnostic method.